The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disordrs or conditions using albumin fusion proteins of the invention.

Images(20)

Claims(25)

1. An albumin fusion protein comprising a member selected from the group consisting of:

(b) a brain derived neurotrophic factor protein and a fragment of the amino acid sequence of SEQ ID NO:18, wherein said fragment has the ability to prolong the shelf-life of the brain derived neurotrophic factor protein compared to the shelf-life of the brain derived neurotrophic factor protein in an unfused state;

(c) a brain derived neurotrophic factor protein and a fragment of the amino acid sequence of SEQ ID NO: 18, wherein said fragment has the ability to prolong the shelf-life of the brain derived growth factor protein compared to the shelf-life of the brain derived neurotrophic factor protein in an unfused state, and further wherein the said fragment comprises amino acid residues 1-387 of SEQ ID NO:18;

(d) a fragment of a brain derived neurotrophic factor protein and albumin comprising the amino acid sequence of SEQ ID NO: 18, wherein said fragment has a biological activity of the brain derived neurotrophic factor protein;

(e) a brain derived neurotrophic factor protein, or fragment thereof and albumin, or fragment thereof, of (a) to (d), wherein the brain derived neurotrophic factor protein or fragment thereof, is fused to the N-terminus of albumin or the N-terminus of the fragment of albumin;

(f) a brain derived neurotrophic factor protein or fragment thereof, and albumin or fragment thereof, of (a) to (d), wherein the brain derived neurotrophic factor protein or fragment thereof, is fused to the C-terminus of albumin, or the C-terminus of the fragment of albumin;

(g) a brain derived neurotrophic factor protein or fragment thereof, and albumin or fragment thereof, of (a) to (d), wherein the brain derived neurotrophic factor protein or fragment thereof, is fused to the N-terminus and C-terminus of albumin, or the N-terminus and the C-terminus of the fragment of albumin;

(h) a brain derived neurotrophic factor protein or fragment thereof, and albumin or fragment thereof, of (a) to (d), which comprises a first brain derived neurotrophic factor protein or fragment thereof and a second brain derived neurotrophic factor protein or fragment thereof, wherein said first brain derived neurotrophic factor protein or fragment thereof is different from said second brain derived neurotrophic factor protein or fragment thereof;

(i) a brain derived neurotrophic factor protein or fragment thereof, and albumin or fragment thereof, of (a) to (h), wherein the brain derived neurotrophic factor protein or fragment thereof, is separated from the albumin or the fragment of albumin by a linker; and

(j) a brain derived neurotrophic factor protein or fragment thereof, and albumin or fragment thereof, of (a) to (i), wherein the brain derived neurotrophic factor protein or fragment thereof, wherein the albumin fusion protein has the following formula:

R1-L-R2; R2-L-R1; or R1-L-R2-L-R1,

and further wherein R1 is brain derived neurotrophic factor protein or fragment thereof, L is linker, and R2 is albumin comprising the amino acid sequence of SEQ ID NO: 18 or a fragment of albumin.

2. The albumin fusion protein of claim 1, wherein the shelf-life of the albumin fusion protein is greater than the shelf-life of the brain derived neurotrophic factor protein or fragment thereof, in an unfused state.

3. The albumin fusion protein of claim 1, wherein the in vitro biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin, or fragment thereof, is greater than the in vitro biological activity of the brain derived neurotrophic factor protein or fragment thereof, in an unfused state.

4. The albumin fusion protein of claim 1, wherein the in viva biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin, or fragment thereof, is greater than the in viva biological activity of the brain derived neurotrophic factor protein or fragment thereof, in an unfused state.

6. An albumin fusion protein comprising a brain derived neurotrophic factor protein or fragment thereof, inserted into an albumin, or fragment thereof, comprising an amino acid sequence selected from the group consisting of:

(a) amino acids residues 54 to 61 of SEQ ID NO:18;

(b) amino acids residues 76 to 89 of SEQ ID NO:18;

(c) amino acids residues 92 to 100 of SEQ ID NO:18;

(d) amino acids residues 170 to 176 of SEQ ID NO:18;

(e) amino acids residues 247 to 252 of SEQ ID NO:18;

(f) amino acids residues 266 to 277 of SEQ ID NO:18;

(g) amino acids residues 280 to 288 of SEQ ID NO:18;

(h) amino acids residues 362 to 368 of SEQ ID NO:18;

(i) amino acids residues 439 to 447 of SEQ ID NO:18;

(j) amino acids residues 462 to 475 of SEQ ID NO:18;

(k) amino acids residues 478 to 486 of SEQ ID NO:18; and

(l) amino acids residues 560 to 566 of SEQ ID NO:18.

7. The albumin fusion protein of claim 5, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the shelf-life of the brain derived neurotrophic factor protein or fragment thereof, as compared to the shelf-life of the brain derived neurotrophic factor protein or fragment, in an unfused state.

8. The albumin fusion protein of claim 6, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the shelf-life of the brain derived neurotrophic factor protein or fragment thereof, as compared to the shelf-life of the brain derived neurotrophic factor protein or fragment, in an unfused state.

9. The albumin fusion protein of claim 5, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the in vitro biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin as compared to the in vitro biological activity of the brain derived neurotrophic factor protein or fragment, in an unfused state.

10. The albumin fusion protein of claim 6, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the in vitro biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin as compared to the in vitro biological activity of the brain derived neurotrophic factor protein or fragment, in an unfused state.

11. The albumin fusion protein of claim 5, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the in vivo biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin as compared to the in vivo biological activity of the brain derived growth factor protein or fragment, in an unfused state.

12. The albumin fusion protein of claim 6, wherein said albumin fusion protein comprises a fragment of albumin sufficient to prolong the in vivo biological activity of the brain derived neurotrophic factor protein or fragment thereof, fused to albumin as compared to the in vivo biological activity of the brain derived neurotrophic factor protein or fragment, in an unfused state.

13. The albumin fusion protein of any of claims 1-12, which is non-glycosylated.

14. The albumin fusion protein of any of claims 1-12, which is expressed in yeast.

15. The albumin fusion protein of any of claim 14, wherein the yeast is glycosylation deficient.

16. The albumin fusion protein of any of claim 14, wherein the yeast is glycosylation and protease deficient.

17. The albumin fusion protein of any of claims 1-12, which is expressed by a mammalian cell.

18. The albumin fusion protein of any of claims 1-12, wherein the albumin fusion protein is expressed by a mammalian cell in culture.

19. The albumin fusion protein of any one of claims 1-12, wherein the albumin fusion protein further comprises a secretion leader sequence.

20. A composition comprising the albumin fusion protein of any one of claims 1-12 and a pharmaceutically acceptable carrier.

21. A kit comprising the composition of claim 20.

22. A method of extending the shelf-life of a brain derived neurotrophic factor protein or fragment thereof, comprising the step of fusing the brain derived neurotrophic factor protein or fragment thereof, to albumin, or fragment thereof, sufficient to extend the shelf-life of the brain derived neurotrophic factor protein, or fragment thereof, compared to the shelf-life of the brain derived neurotrophic factor protein, or fragment thereof in an unfused state.

23. A nucleic acid molecule comprising a polynucleotide sequence encoding the albumin fusion protein of any one of claims 1-12.

24. A vector comprising, the nucleic acid molecule of claim 23.

25. A host cell comprising the nucleic acid molecule of claim 24.

Description

This application claims the benefit of priority under 35 U.S.C. § 119(e) based on the following U.S. provisional applications: 60/229,358 filed on Apr. 12, 2000; 60/199,384 filed on Apr. 25, 2000; and 60/256,931 filed on Dec. 21, 2000. Each of the provisional applications is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates generally to Therapeutic proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or fragments or variants of albumin. The invention further relates to Therapeutic proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or fragments or variants of albumin, that exhibit extended shelf-life and/or extended or therapeutic activity in solution. These fusion proteins are herein collectively referred to as “albumin fusion proteins of the invention.” The invention encompasses therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention using these nucleic acids, vectors, and/or host cells.

The invention is also directed to methods of in vitro stabilizing a Therapeutic protein via fusion or conjugation of the Therapeutic protein to albumin or fragments or variants of albumin.

Human serum albumin (HSA, or HA), a protein of 585 amino acids in its mature form (as shown in FIG. 15 or in SEQ ID NO: 18), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.

The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a component of an albumin fusion protein as a carrier for various proteins has been suggested in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of HA for fusions to polypeptides has also been proposed (EP 399 666). Fusion of albumin to the Therapeutic protein may be achieved by genetic manipulation, such that the DNA coding for HA, or a fragment thereof, is joined to the DNA coding for the Therapeutic protein. A suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to express a fusion polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo e.g. from a transgenic organism.

Therapeutic proteins in their native state or when recombinantly produced, such as interferons and growth hormones, are typically labile molecules exhibiting short shelf-lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that many of the molecules must be lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside of the hospital environment. Many protein and peptide drugs also require the addition of high concentrations of other protein such as albumin to reduce or prevent loss of protein due to binding to the container. This is a major concern with respect to proteins such as IFN. For this reason, many Therapeutic proteins are formulated in combination with large proportion of albumin carrier molecule (100-1000 fold excess), though this is an undesirable and expensive feature of the formulation.

Few practical solutions to the storage problems of labile protein molecules have been proposed. Accordingly, there is a need for stabilized, long lasting formulations of proteinaceous therapeutic molecules that are easily dispensed, preferably with a simple formulation requiring minimal post-storage manipulation.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that Therapeutic proteins may be stabilized to extend the shelf-life, and/or to retain the Therapeutic protein's activity for extended periods of time in solution, in vitro and/or in vivo, by genetically or chemically fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to stabilize the protein and/or its activity. In addition it has been determined that the use of albumin-fusion proteins or albumin conjugated proteins may reduce the need to formulate protein solutions with large excesses of carrier proteins (such as albumin, unfused) to prevent loss of Therapeutic proteins due to factors such as binding to the container.

The present invention encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or a fragment (portion) or variant of albumin. The present invention also encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or a fragment (portion) or variant of albumin, that is sufficient to prolong the shelf life of the Therapeutic protein, and/or stabilize the Therapeutic protein and/or its activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells.

The invention also encompasses pharmaceutical formulations comprising an albumin fusion protein of the invention and a pharmaceutically acceptable diluent or carrier. Such formulations may be in a kit or container. Such kit or container may be packaged with instructions pertaining to the extended shelf life of the Therapeutic protein. Such formulations may be used in methods of treating, preventing, ameliotationg or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.

In other embodiments, the present invention encompasses methods of preventing treating, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indication Y” column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein (or fragment or variant thereof) disclosed in the “Therapeutic Protein X” column of Table 1 (in the same row as the disease or disorder to be treated is listed in the “Preferred Indication Y” column of Table 1) in an amount effective to treat prevent or ameliorate the disease or disorder.

In another embodiment, the invention includes a method of extending the shelf life of a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) comprising the step of fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to extend the shelf-life of the Therapeutic protein. In a preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the fragment or variant of albumin. In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment or variant of albumin, via recombinant DNA technology or genetic engineering.

In another embodiment, the invention includes a method of stabilizing a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) in solution, comprising the step of fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to stabilize the Therapeutic protein. In a preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the fragment or variant of albumin. In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment or variant of albumin, via recombinant DNA technology or genetic engineering.

The present invention further includes transgenic organisms modified to contain the nucleic acid molecules of the invention, preferably modified to express the albumin fusion proteins encoded by the nucleic acid molecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the extended shelf-life of an HA fusion protein in terms of the biological activity (Nb2 cell proliferation) of HA-hGH remaining after incubation in cell culture media for up to 5 weeks at 37° C. Under these conditions, hGH has no observed activity by week 2.

FIG. 2 depicts the extended shelf-life of an HA fusion protein in terms of the stable biological activity (Nb2 cell proliferation) of HA-hGH remaining after incubation in cell culture media for up to 3 weeks at 4, 37, or 50° C. Data is normalized to the biological activity of hGH at time zero.

FIGS. 3A and 3B compare the biological activity of HA-hGH with hGH in the Nb2 cell proliferation assay. FIG. 3A shows proliferation after 24 hours of incubation with various concentrations of hGH or the albumin fusion protein, and FIG. 3B shows proliferation after 48 hours of incubation with various concentrations of hGH or the albumin fusion protein.

FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as the base vector into which polynucleotides encoding the Therapeutic proteins (including polypeptides and fragments and variants thereof) may be cloned to form HA-fusions. Plasmid Map key: PRB1p: PRB1 S. cerevisiae promoter; FL: Fusion leader sequence; rHA: cDNA encoding HA ADH1t: ADH1 S. cerevisiae terminator; T3: T3 sequencing primer site; T7: T7 sequencing primer site; Amp R: β-lactamase gene; ori: origin of replication. Please note that in the provisional applications to which this application claims priority, the plasmid in FIG. 4 was labeled pPPC0006, instead of pPPC0005. In addition the drawing of this plasmid did not show certain pertinent restriction sites in this vector. Thus in the present application, the drawing is labeled pPPC0005 and more restriction sites of the same vector are shown.

FIG. 5 compares the recovery of vial-stored HA-IFN solutions of various concentrations with a stock solution after 48 or 72 hours of storage.

FIG. 6 compares the activity of an HA-α-IFN fusion protein after administration to monkeys via IV or SC.

FIG. 7 describes the bioavailability and stability of an HA-α-IFN fusion protein.

FIG. 8 is a map of an expression vector for the production of HA-α-IFN.

FIG. 9 shows the location of loops in HA.

FIG. 10 is an example of the modification of an HA loop.

FIG. 11 is a representation of the HA loops.

FIG. 12 shows the HA loop IV.

FIG. 13 shows the tertiary structure of HA.

FIG. 14 shows an example of a scFv-HA fusion

FIG. 15 shows the amino acid sequence of the mature form of human albumin (SEQ ID NO:18) and a polynucleotide encoding it (SEQ ID NO:17).

DETAILED DESCRIPTION

As described above, the present invention is based, in part, on the discovery that a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) may be stabilized to extend the shelf-life and/or retain the Therapeutic protein's activity for extended periods of time in solution (or in a pharmaceutical composition) in vitro and/or in vivo, by genetically fusing or chemically conjugating the Therapeutic protein, polypeptide or peptide to all or a portion of albumin sufficient to stabilize the protein and its activity.

The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. As used herein, “albumin fusion protein” refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or chemical conjugation to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may be referred to as a “portion”, “region” or “moiety” of the albumin fusion protein (e.g., a “Therapeutic protein portion” or an “albumin protein portion”).

In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.

In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein. In a further preferred embodiment, the Therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the Therapeutic protein. In an alternative embodiment, the Therapeutic protein portion of the albumin fusion protein is the active form of the Therapeutic protien.

In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin.

Therapeutic Proteins

As stated above, an albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion or chemical conjugation.

As used herein, “Therapeutic protein” refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities. Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics. (The terms peptides, proteins, and polypeptides are used interchangeably herein.) It is specifically contemplated that the term “Therapeutic protein” encompasses antibodies and fragments and variants thereof. Thus an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an antibody. Additionally, the term “Therapeutic protein” may refer to the endogenous or naturally occurring correlate of a Therapeutic protein.

By a polypeptide displaying a “therapeutic activity” or a protein that is “therapeutically active” is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a Therapeutic protein such as one or more of the Therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a “Therapeutic protein” is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder. As a non-limiting example, a “Therapeutic protein” may be one that binds specifically to a particular cell type (normal (e.g., lymphocytes) or abnormal e.g., (cancer cells)) and therefore may be used to target a compound (drug, or cytotoxic agent) to that cell type specifically.

In another non-limiting example, a “Therapeutic protein” is a protein that has a biological activity, and in particular, a biological activity that is useful for treating preventing or ameliorating a disease. A non-inclusive list of biological activities that may be possessed by a Therapeutic protein includes, enhancing the immune response, promoting angiogenesis, inhibiting angiogenesis, regulating hematopoietic functions, stimulating nerve growth, enhancing an immune response, inhibiting an immune response, or any one or more of the biological activities described in the “Biological Activities” section below.

As used herein, “therapeutic activity” or “activity” may refer to an activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms. Therapeutic activity may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture. As an example, when hGH is the Therapeutic protein, the effects of hGH on cell proliferation as described in Example 1 may be used as the endpoint for which therapeutic activity is measured. Such in vitro or cell culture assays are commonly available for many Therapeutic proteins as described in the art.

Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, such as cell surface and secretory proteins, are often modified by the attachment of one or more oligosaccharide groups. The modification, referred to as glycosylation, can dramatically affect the physical properties of proteins and can be important in protein stability, secretion, and localization. Glycosylation occurs at specific locations along the polypeptide backbone. There are usually two major types of glycosylation: glycosylation characterized by O-linked oligosaccharides, which are attached to serine or threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where X can be any amino acid except proline. N-acetylneuramic acid (also known as sialic acid) is usually the terminal residue of both N-linked and O-linked oligosaccharides. Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites. Glycosylation isomers are also common at the same site within a given cell type.

Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, as well as analogs and variants thereof, may be modified so that glycosylation at one or more sites is altered as a result of manipulation(s) of their nucleic acid sequence, by the host cell in which they are expressed, or due to other conditions of their expression. For example, glycosylation isomers may be produced by abolishing or introducing glycosylation sites, e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or unglycosylated recombinant proteins may be produced by expressing the proteins in host cells that will not glycosylate them, e.g. in E. coli or glycosylation-deficient yeast. These approaches are described in more detail below and are known in the art.

Additional Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, one or more of the Therapeutic proteins or peptides disclosed in the “Therapeutic Protein X” column of Table 1, or fragment or variable thereof.

Table 1 provides a non-exhaustive list of Therapeutic proteins that correspond to a Therapeutic protein portion of an albumin fusion protein of the invention. The “Therapeutic Protein X” column discloses Therapeutic protein molecules followed by parentheses containing scientific and brand names that comprise, or alternatively consist of, that Therapeutic protein molecule or a fragment or variant thereof. “Therapeutic protein X” as used herein may refer either to an individual Therapeutic protein molecule (as defined by the amino acid sequence obtainable from the CAS and Genbank accession numbers), or to the entire group of Therapeutic proteins associated with a given Therapeutic protein molecule disclosed in this column. The “Exemplary Identifier” column provides Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or Genbank Accession Numbers ((e.g., Locus ID, NP_XXXXX (Reference Sequence Protein), and XP_XXXXX (Model Protein) identifiers available through the national Center for Biotechnology Information (NCBI) webpage at www.ncbi.nlm.nih.gov) that correspond to entries in the CAS Registry or Genbank database which contain an amino acid sequence of the Therapeutic Protein Molecule or of a fragment or variant of the Therapeutic Protein Molecule. In addition GenSeq Accession numbers and/or journal publication citations are given to identify the exemplary amino acid sequence for some polypeptides. The summary pages associated with each of these CAS and Genbank and GenSeq Accession Numbers as well as the cited journal publications (e.g., PubMed ID number (PMID)) are each incorporated by reference in their entireties, particularly with respect to the amino acid sequences described therein. The “PCT/Patent Reference” column provides U.S. Patent numbers, or PCT International Publication Numbers corresponding to patents and/or published patent applications that describe the Therapeutic protein molecule. Each of the patents and/or published patent applications cited in the “PCT/Patent Reference” column are herein incorporated by reference in their entireties. In particular, the amino acid sequences of the specified polypeptide set forth in the sequence listing of each cited “PCT/Patent Reference”, the variants of these amino acid sequences (mutations, fragments, etc.) set forth, for example, in the detailed description of each cited “PCT/Patent Reference”, the therapeutic indications set forth, for example, in the detailed description of each cited “PCT/Patent Reference”, and the activity assays for the specified polypeptide set forth in the detailed description, and more particularly, the examples of each cited “PCT/Patent Reference” are incorporated herein by reference. The “Biological activity” column describes Biological activities associated with the Therapeutic protein molecule. The “Exemplary Activity Assay” column provides references that describe assays which may be used to test the therapeutic and/or biological activity of a Therapeutic protein or an albumin fusion protein of the invention comprising a Therapeutic protein X portion. Each of the references cited in the “Exemplary Activity Assay” column are herein incorporated by reference in their entireties, particularly with respect to the description of the respective activity assay described in the reference (see Methods section, for example) for assaying the corresponding biological activity set forth in the “Biological Activity” column of Table 1. The “Preferred Indication Y” column describes disease, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by Therapeutic protein X or an albumin fusion protein of the invention comprising a Therapeutic protein X portion.

Exemplary

PCT/Patent

Exemplary Activity

Therapeutic Protein X

Identifier

Reference

Biological Activity

Assay

Preferred Indication Y

ABC1

LocusID: 19

WO9837764-A1

ABC-1 is a membrane-associated member

ABC-1 function may be assayed

Atherosclerosis; Tangier

NP_005493

WO9712346-A2

of the superfamily of ATP-

by measuring

Disease;

XP_005567

WO8748797-A1

binding cassette (ABC) transporters.

apolipoprotein-mediated

Hypercholesterolemia

WO200055318-A2

ABC proteins transport various

lipid efflux from cultured

molecules across extra- and intracellular

cells. (J Clin Invest 1999

membranes. This protein is a member of the ABC1

Oct; 104(8): R25-31).

subfamily, the only major ABC subfamily

found exclusively in multicellular eukaryotes. With

cholesterol as its substrate, this protein

functions as a cholesteral efflux pump in

the cellular lipid removal pathway.

Acidic FGF-

LocusID: 2246

WO8705332-A

Binds to and kills cells expressing the

aFGF1-pseudomonas

Cancer; Vascular Disease

Pseudomonas exotoxin

NP_000791

WO9503831-A

Acidic fibroblast growth factor 1

exotoxin chimera function

Fusion protein

Genbank: P11439

US5827826-A

receptor.

may be assayed in vitro

Genbank: K01397

US5849538-A

using a cytotoxicity

US6077692-A

assay. (Proc Natl Acad

US6037329-A

Sci USA 1989 Jun;

AUS6077692-A

86(11): 4215-4219).

Activin (Activin A;

CAS-

EP222491

The Activin A complex, composed of a

Inhibin A activity may be

Fracture; Postmenopausal

EDF, inhibin, beta A)

114949-22-3

WO200017360

homodimer of inhibin beta A subunits,

assayed in vitro by

Osteoporosis; Cancer;

LocusID: 3624

is a pituitary FSH secretion inhibitor,

measuring its

Male Infertility; Female

NP_002183

which has been shown to regulate

differentiation-inducing

Infertility; Sickle Cell Anemia

XP_004832

gonadal stromal cell proliferation

activity toward mouse

negatively and to have tumour-

Friend erythroleukemia

suppressor activity. In addition, serum

(MEL) cells and human

levels of this complex have been shown

K-562 cells (Proc Natl

to reflect the size of granulosa-cell

Acad Sci USA 1988

tumors and can therefore be used as a

Apr; 85(8): 2434-2438);

marker for primary as well as recurrent disease.

or its suppression of the

secretion of pituitary

follicle-stimulating

hormone (Biol Reprod

2000 Sep; 63(3): 865-871).

Activin beta c (inhibin,

LocusID: 3626

DE19511243

Inhibin beta C subunit is similar to the

Inhibin beta C activity

Neurodegenerative

beta c)

NP_005529

WO9602559

inhibin/activin beta A and beta B

may be assayed in vitro

Disorders; Wound

XP_006661

WO9822492

subunits and may be part of a

by measuring its

Healing; Liver

transforming growth factor-beta

differentiation-inducing

Regeneration

superfamily.

activity toward mouse

Friend erythroleukemia (MEL)

cells and human

K-562 cells (Proc Natl Acad

Sci USA 1988

Apr; 85(8): 2434-2438); or its

suppression of the secretion of

pituitary follicle-stimulating

hormone (Biol Reprod 2000

Sep; 63(3): 865-871).

Adenosine deaminase

LocusID: 100

WO200109305

Adenosine deaminase catalyzes the

Adenosine deaminase

Adenosine Deaminase

(Pegademase

NP_000013

WO200050609-A1

hydrolysis of adenosine to inosine.

activity may be assayed

Deficiency In Patients

(pegylated); ADAGEN)

XP_009517

in vitro by measuring

With Severe Combined

purine catabolism (Mol Cell Biol

Immunodeficiency (SCID)

1985 Apr; 5(4):

762-767).

Adiposin (Lipotropin)

CAS-9035-

EP926239-A2

Adiposin is produced by the sequential

Adiposin function may be

Obesity; Cancer

55-6

WO200053755-A2

cleavage of the precursor protein pre-pro-

assayed in vitro by

Genbank: 223277

WO200037615-A1

opiomelanocortin (POMC). Adiposin

measuring cAMP

Genbank: 223281

plays a key role in the regulation of food

accumulation in adiposin

intake via activation of the brain

stimulated mouse

melanocortin-4-receptor.

melanoma cells. (Hum

Mol Genet 1995 Feb;

4(2): 223-230).

Agouti signal protein

LocusID: 434

WO9700892-A2

Agouti Signaling Protein (ASP),

ASP function may be

Pigmentation Disorders;

NP_001663

WO200044898-A2

encodes a 132 amino acid protein, the

assayed in vitro by

Obesity

XP_009476

mRNA for which is expressed in testis,

measuring inhibition of

ovary, and heart, and at lower levels in

alpha-MSH-stimulated

liver, kidney, and foreskin. ASP

cAMP accumulation in

interacts with the extension gene (which

mouse melanoma cells.

encodes the melanocyte receptor for

(Hum Mol Genet 1995

alpha-melanocyte stimulating hormone

Feb; 4(2): 223-230).

[alpha-MSH]), and expression of ASP in

cell culture blocks the alpha-MSH-stimulated

accumulation of cAMP in mouse melanoma cells.

AI 100 (Bovine Myelin;

Genbank: MB

WO8912459

Myelin (myelin basic protein) is the

Myelin may be assayed

Multiple Sclerosis

Myloral)

BOB

WO9218150

constituent of the myelin sheath and

in vitro by measurement

US5763585

plays an important role in efficient nerve

of its phosphorylation by

US5284935

function.

MAP kinase (J

Neurochem 1999 Sep; 73(3):

1090-1097); or its effect on synaptic

transmission between neurons

(Eur Neurol. 1988; 28(2): 57-63).

AI 101 (Recombinant

LocusID: 4155

WO9634622-A1

Myelin basic protein is a constituent of

Myelin Basic Protein

Multiple Sclerosis

Human Myelin Basic

NP_002376

WO9634622-A1

myelin and plays an important role in

may be assayed in vitro

Protein)

XP_012271

WO9218150-A

efficient nerve function.

by measurement of its

WO8912459-A

phosphorylation by MAP

kinase (J Neurochem

1999 Sep; 73(3): 1090-1097);

or its effect on

synaptic transmission

between neurons (Eur

Neurol. 1988; 28(2): 57-63).

AI 200 (type II collagen;

LocusID: 1280

WO9535373-A2

Alpha 1 subunit of type II collagen is a

Collagen function may be

Juvenile Rheumatoid

Colloral)

NP_001834

WO9637776-A1

member of the fibrillar collagen family of

measured using an in

Arthritis; Rheumatoid

XP_012271

WO9830695-A2

extracellular matrix (ECM) proteins.

vitro collagen fibril

Arthritis

stability assay (Cell Mol

Life Sci 2000 May;

57(5): 859-863); or an in

vitro cell adhesion assay (J Cell

Biochem Oct. 1, 1997;

67(1): 75-83).

Alpha glucosidase (Acid

LocusID: 2548

US5763252-A

Hydrolysis of alpha1-6 linkages in

Hydrolysis of

Pompe's Disease

alpha-glucosidase;

NP_000143

EP394538-A

glycogen; debranching enzyme;

chromogenic artificial

Pompase)

XP_012708

WO9851325

degradation of starch in muscle, liver and

substrate p-Nitrophenyl a-

other tissues

D-glucoside.

Bergmeyer, H. U. (ed)

Methods of Enzymatic Analysis,

Second English Edition, 1, 459 (1974)

Alpha-galactosidase

LocusID: 2717

US5356804-A

Hydrolytic cleavage of galactose residues

Hydrolysis of

Fabry's Disease

(Arginosuccinate lyase;

NP_000160

WO9412628-A

from the glycosphingolipid

chromogenic artificial

Beta-glucosidase; x-

XP_010108

WO9507088-A

galactosylgalactosylglucosylceramide

substrate p-Nitrophenyl a-

galactosidase A;

WO9011353-A

D-galactosidase to p-

agalsidase alpha; CC-

US5658567

Nitrophenol and D-

galactosidase; Fabrazyme;

US5179023

galactose- Rietra et

Replagal)

WO9811206

al., (1975) “Properties

of the residual alpha-

galactosidase activity in the tissues of

a Fabry hemizygote”. Clin

Chim Acta; 62(3): 401-13

Alpha-L iduronidase

LocusID: 3425

WO9958691-A2

Iduronidase is a lysosomal enzyme that

Hydrolysis of the

Mucopolysaccharidosis I;

(Alronidase; Laronidase;

NP_000194

WO9710353-A1

participates in the degradation of

substrate 4-

Mucopolysaccharidosis Ii;

Iduronato-2-sulfatase;

WO9310244-A

dermatan sulfate and heparan sulfate.

methylumbelliferyl alpha-

Hurler Syndrome; Hurler-

Aldurazyme; Replidur)

L-iduronide is followed

Scheie Syndrome; Scheie

in a fluorometric assay.

Syndrome; Hunter

Hopwood et al. (1979),

Syndrome

Clin Chim Acta.; 92: 257-65.

Other assays are found in

Thompson

(1978) “Substrates for the assay of

alpha-L-iduronidase”. Clin

Chim Acta; 89(3): 435-46

Angiopoietin 1

NP_001137

WO200064946-A2

Angiopoeitin 1 is a growth factor

Angiopoeitin 1 activity

Inflammatory Disorders;

WO200002584-A2

involved in the control of angiogenesis

may be assayed in vitro

Cardiovascular Disorders

through the Tie receptors.

using a capillary

sprouting assay. (Curr

Biol Apr. 23, 1998;

8(9): 529-532).

Angiopoietin 2

Genbank: BAA95590

WO200064946

Angiopoeitin 2 is a growth factor

Angiopoeitin 2 activity

Cancer

WO9631598

involved in the control of angiogenesis

may be assayed in vitro

through the Tie receptors.

using a capillary

sprouting assay. (Curr Biol

Apr. 23, 1998;

8(9): 529-532).

Angiostatin

CAS-86090-

WO9013640-A

Angiostatin represents an internal

Endothelial proliferation

Solid Tumors

(Angiostatin)

08-6

US5200340-A

fragment of plasminogen (containing the

assay: Kringle domains of

LocusID: 5340

WO9108297-A

first four kringle structures). Individual

Human Angiostatin. Cao

NP_000292

US5648254-A

or combined kringle structures of

et al (1996) J. Biol.

XP_004371

US6057122-A

angiostatin have been shown to inhibit

Chem. 271 29461-29467

effects of on capillary endothelial cell proliferation.

Anti-dorsalizing

Genbank: AAC59736

US5693779

ADMP is related to human Bone

ADMP activity may be

Cancer

morphogenetic protein-1

Genbank: AAD52011

Morphogenetic Protein-3. Its expression

assayed in vitro by

(ADMP)

peaks during gastrulation and results in

measuring its ability to

dose-dependent suppression of dorsal

downregulate the

and anterior structures.

dorsalizing factors

noggin, goosecoid or

follistatin. (Development 1995 Dec;

121(12): 4293-4301).

APO2 Ligand (TRAIL)

LocusID: 8743

WO9733899

Type II glycoprotein of the tumor

Apoptosis assay:

Cancer

NP_003801

WO9701633

necrosis factor ligand superfamily;

TRAIL-R2: a novel

XP_003200

EP870827

mediates cell death. TRAIL can induce

apoptosis-mediating

US5763223

apoptosis in a wide variety of

receptor for TRAIL.

US604608

transformed cell lines of diverse

Walczak et al. (1996)

lineage, but does not appear to kill

EMBOJ 16: 5386-5397

normal cells.

Arresten

Genbank: AF72630

WO8903392

Arresten functions as an anti-angiogenic

Arresten function may be

Cancer; Solid Tumors

US5114840

molecule by inhibiting endothelial cell

assayed in vitro by

US5593900

proliferation, migration, tube formation,

measuring its ability to

and Matrigel neovascularization.

inhibit endothelial cell

proliferation, migration,

tube formation, and

Matrigel

neovascularization.

(Cancer Res May 1, 2000;

60(9): 2520-6).

Arylsulfatase B (BM

LocusID: 411

US6153188-A

The arylsulfatase B homodimer

Arylsulfatase B activity

Mucopolysaccharidosis

102; recombinant human

NP_000037

WO9946281-A2

hydrolyzes sulfate groups of N-Acetyl-D-

may be assayed by in

VI

N-acetylgalactosamine-4-

Genbank: AAB19988

WO9950453-A1

galactosamine, chondriotin sulfate, and

vitro measurement of

sulfatase; rhASB)

Genbank: CAA51272

dermatan sulfate. The protein is targeted

hydrolysis of sulfates of

Genbank: AAA51784

to the lysozyme. Defects in this gene

N-Acetyl-D-

Genbank: AAA51779

cause Maroteaux-Lamy syndrome.

galactosamine. (J Biol

Chem Feb. 25, 1990;

265(6): 3374-3381).

Asparginase

CAS-

EP211639-A

Asparaginase is the enzyme responsible

Asparaginase activity

Acute Lymphoblastic

(Pegaspargase;

130167-69-0

WO9202616-A

for hydrolysis of L-Asparagine.

may be assayed in vitro

Leukemia

Asparaginase macrogol;

Genbank: CAA01168

EP811687-A2

using an asparaginase

PEG-asparaginase;

enzymatic assay. (Anal

PEGLA; Oncaspar)

Biochem Apr. 10, 2000;

280(1): 42-45).

Bactericidal permeability

LocusID: 671

WO9418323-A

A membrane-associated protein, similar

rBPI activity may be

Ocular Inflammation;

increasing protein 21

NP_001716

WO9420128-A

to LBP, CETP, and PLTP, with

assayed in vitro using an

Cystic Fibrosis;

(rBPI 21; I-PREX;

AAA51841

US5439807-A

bactericidal activity.

antibacterial assay. (J

Haemorrhagic Trauma;

Opebecan; Neuprex)

US5523288-A

Biol Chem Nov. 5, 1987;

Intra-Abdominal

262(31): 14891-14894).

Infections; Meningococcal

Infection;

Meningococcemia; Otitis

Media; Partial

Hepatectomy;

Toxoplasmosis

BDNF (Brain-derived

LocusID: 627

WO9103568-A

Brain-derived neurotrophic factor

Neuronal growth and

Amyotrophic Lateral

neurotrophic factor)

NP_001700

JP05317049-A

promotes the survival of neuronal

synaptic activity assays:

Sclerosis;

XP_006027

US5438121-A

populations that are all located either in

BDNF

Neurodegeneration

WO9310150-A

the central nervous

enhances neuronal growth

WO9103568-A

system or directly connected to it.

and synaptic activity in

US6121235-A

hippocampal cell

WO9202620-A

cultures. Bartrupet al

(1997) Neuroreport

1; 8(17): 3791-4

B-glucocerebrosidase

CAS-

US7137796-A

glucocerebrosidase (beta-glucosidase)

Daniels L B, Glew R H,

Gaucher's Disease

(Alglucerase (human);

143003-46-7

US5879680-A

catalyzes the removal of glucose from

Radin N S, Vunnam R R.

Imiglucerase; PEG-

CAS-

US6074864

glucosylceramide to form ceramide

A revised

glucocerebrosidase;

154248-97-2

US5549892-A

fluorometric assay for

Ceredase; Cerezyme;

LocusID: 2629

WO9710353

Gaucher's disease using

Selezyme; Lysodase)

NP_000148

conduritol-beta-epoxide

XP_002191

with liver as the source of

Beta-glucosidase. Clin

Chim Acta. Sep. 25, 1980;

106(2): 155-63.

Johnson W G, Gal

A E, Miranda A F,

Pentchev P G.

Diagnosis of adult

Gaucher disease: use of a

new chromogenic substrate,

2-hexadecanoylamino-4-

nitrophenyl-beta-D-

glucopyranoside, in

cultured skin fibroblasts.

Clin Chim Acta. Mar. 14, 1980;

102(1): 91-7.

BMP-2 (Bone

CAS-

US5013649-A

BMP2 belongs to the transforming

Wang, E. A.; Rosen, V.;

Bone Regeneration; Bone

Morphogenetic Protein

192509-82-3

US5166058-A

growth factor-beta (TGFB) superfamily.

D'Alessandro, J. S.;

And Tissue Repair;

2; Bone-related protein)

LocusID: 650

WO9309229-A

Bone morphogenic protein induces bone

Bauduy, M.; Cordes, P.;

Cancer

NP_001191

WO9403600-A

formation. BMP2 is a candidate gene

Harada, T.; Israel, D. I.;

XP_009629

US6150328-A

for the autosomal dominant disease of

Hewick, R. M.; Kerns,

WO8800205-A

fibrodysplasia (myositis) ossificans

K. M.; LaPan, P.;

WO9118047-A

progressiva.

Luxenberg, D. P.;

McQuaid, D.;

Moutsatsos, I. K.; Nove,

J.; Wozney, J. M.:

Recombinant human

bone morphogenetic protein

induces bone

formation. Proc. Nat.

Acad. Sci. 87: 2220-2224,

1990.

Botulinum toxin-

Genbank: A43503

EP492447-A

Botulinum toxins is selectively targeted

BT-SD function may be

Stroke; Trauma

superoxide dismutase

Genbank: BAB03518

EP676472-A1

to cholinergic nerve terminals.

assayed in vitro by using

(BT-SD)

CAS-9016-

US5540911-A

Superoxide dismutase is an intracellular

a superoxide dismutase

01-7

US5683864-A

protein which catalyzes dismutation of

assay. (Nucleic Acids Res

LocusID: 6647

US5688688-A

superoxide to oxygen and hydrogen

Mar. 25, 1985;

LocusID: 6648

US5712087-A

peroxide. Botulinum toxin targeting of

13(6): 2017-34).

LocusID: 6649

US5780024-A

superoxide dismutase to specific neurons

NP_000445

US5849290-A

may aid in the treatment of diseases

NP_000627

US6107070-A

characterized by production of

NP_003093

WO8701387-A

superoxide in those neurons.

XP_009723

WO9009434-A

XP_004242

WO9010694-A

XP_003578

WO9104315-A

WO9414950-A

WO9419493-A

BRCA1 (BRCA1 tumor

Lcous ID: 672

WO9957132

BRCA1, a tumor suppressor in human

BRCA1 activity may be

Cancer; Breast Cancer;

suppressor protein)

NP_006759

breast cancer cells, is a nuclear

assayed by measuring

Prostrate Cancer; Ovarian

XP_007013

phosphoprotein which associates with

alterations in expression

Cancer

RNA polymerase II holoenzyme.

of p21WAF1/CIP1.

BRCA1 functions as a transcriptional

(Oncogene Jun. 11, 1998;

regulator and is also a granin-like

16(23): 3069-82).

protein that functions as a secreted

growth inhibitory protein.

BRCA2

Locus ID: 675

WO9909164-A1

BRCA2, a tumor suppressor in human

BRCA2 activity may be

Cancer; Breast Cancer;

NP_000050

WO9722689-A1

breast cancer cells, is a nuclear

assayed by measuring

Ovarian Cancer

XP_007138

WO9730108-A1

phosphoprotein which associates with

alterations in expression

RNA polymerase II holoenzyme.

of p21 WAF1/CIP1.

BRCA2 exists in a dimeric complex

(Oncogene Jun. 11, 1998;

with BRCA1, functions as a

16(23): 3069-82).

transcriptional regulator, and is also a granin-like protein

that functions as a secreted growth inhibitory protein.

Calcitonin gene-related

CAS-83652-

EP188400-A

CGRP is a potent vasodilator, and a

The vasodilatory activity

Angina Pectoris;

peptide (CGRP)

28-2

EP367463-A

regulator of endothelial and osteoblast

of CGRP can be assayed

Arrythmias; Heart

CAS-83652-

EP70675-A

cell proliferation. Additional effects of

using the aortic ring

Failure; Hypertension;

28-2

EP821061-A2

CGRP include reduced gastric secretion,

vasodilation assay

Postmenopausal

Locus ID: 796

EP95351-A

increased body temperature, anorexic

described in Pharmacol

Osteoporosis; Raynaud's

Locus ID: 797

JP05255391-A

effects, and positive inotropic and

Res. 1999

Disease; Subarachnoid

Locus

US5858978-A

chronotropic effects on the heart.

Mar; 39(3): 217-20.

Haemorrhage

ID: 27297

WO9741223-A1

Endothelial and

NP_001732

WO9415962-A

osteoblast cell

NP_000719

WO9734922-A1

proliferation activities can

NP_055293

WO9803534-A1

be measured in vitro (Eur

XP_006209

J Pharmacol. Dec. 15, 2000;

XP_006016

409(3): 273-8; Proc

XP_004758

Natl Acad Sci U.S.A.

1990 May; 87(9): 3299-303)

Calreticulin

Locus ID: 811

WO9636643

Calreticulin is a multifunctional protein

Calreticulin activity can

Cancer; Wound Healing

NP_004334

WO9907406

that acts as a major Ca(2+)-binding

be measured in vitro

XP_009055

(storage) protein in the lumen of the

using calcium imaging

Genbank: AH02500

endoplasmic reticulum. Calreticulin can

assays (Cell. Sep. 8, 1995;

act as an important modulator of the

82(5): 765-71).

regulation of gene transcription by

nuclear hormone receptors.

CD4 (CD4; rCD4;

Locus ID: 920

WO9118618-A

CD4 is a T-cell surface glycoprotein

CD4 function may be

Hiv Infection

recombinant CD4;

NP_000607

US5126433-A

which plays roles in cell-cell

assayed in vitro by

recombinant soluble

XP_006966

WO8801304-A

interactions in signal transduction. CD4

measuring gp120 binding

CD4; recombinant

US5958678-A

binds gp120 of HIV and is a cell surface

(Viral Immunol 2000;

soluble human CD4;

US6093539-A

receptor for HIV entry.

13(4): 547-554); or altered

rST4; soluble CD4;

US5411861-A

monocyte responses to

soluble T4; T4 receptor)

US5955264-A

cytokines following

gp120 binding (J Immunol

Oct. 15, 1998; 161(8): 4309-4317).

CD40 ligand (CD40-L;

LocusID: 959

WO9308207-A

CD40-L is expressed on the surface of T

Hollenbaugh D,

Epithelial Solid Tumors;

Avrend)

NP_000065

WO9410308-A

cells. It regulates B cell function by

Grosmaire L S, Kullas

Head And Neck Cancer;

XP_010367

US5674492-A

engaging CD40 on the B cell surface. A

C D, Chalupny N J,

Immunodeficiency

US5817516-A

defect in this gene results in inability of

Braesch-Andersen S,

Disorders; Non-Hodgkin's

US5716805-A

immunoglobulin class switch and is

Noelle R J, Stamenkovic

Lymphoma; Renal

WO9517202-A1

associated with hyper IgM syndrome.

I, Ledbetter J A, Aruffo A.

Cancer; Renal Cell

WO9308207-A

The human T cell

Carcinoma; Solid

antigen gp39, a member

Tumors; Viral Infections

of the TNF gene family,

is a ligand for the CD40 receptor:

expression of a

soluble form of gp39 with

B cell co-stimulatory activity.

EMBO J. 1992

Dec; 11(12): 4313-21.

Chemokine Binding

LocusID:

WO9906561-A2

Chemokine binding proteins are

Chemokine binding

Transplant Rejection;

Proteins (CBP 1; CPB

1238

WO9623068-A1

involved in chemokine-mediated

proteins can be assayed

Cardiovascular Disease;

2)

NP_001287

WO9947697-A1

signaling.

using receptor binding

Rheumatoid Arthritis;

XP_003126

WO9412635-A

assays (J Biol Chem

Inflammatory Disorders;

LocusID: 1241

US5759804-A

May 9, 1997;

Immune System

NP_000743

US6107475-A

272(19): 12495-12504)

Disorders

XP_007314

WO9728188-A1

Ciliary neurotrophic

LocusID: 1270

WO9104316-A

CNTF promotes the differentiation and

CNTF may be assayed in

Amyotrophic Lateral

factor (CNTF; Axokine)

NP_000605

WO9302206-A

survival of a wide range of cell types in

vitro using neuronal

Sclerosis; Diabetic

XP_006012

WO9307270-A

the nervous system

proliferation and survival

Neuropathies;

WO9311253-A

assays (EMBO J. Apr. 2, 2001;

Huntington's Disease;

US5846935-A

20(7) 1692-1703)

Obesity; Retinal

US5846935-A

Disorders; Type 2

Diabetes Mellitus

Contortrostatin

CAS-

WO200018421-A1

Contortrostatin is a unique dimeric

Contortrostatin activity

Cancer Metasteses;

(Agkistrodon contortrix

153858-68-5

EP323722-A

disintegrin which antagonizes integrins

may be measured in vitro

Thrombosis; Stroke

contortrix (southern

Genbank: AAF65171

WO9846771-A2

alphaIIbbeta3, alpha5beta1, alphavbeta3,

by measuring binding to

copperhead snake);

and alphavbeta5, and thereby inhibits

integrins alphavbeta3 and

Disintegrin)

platelet aggregation and disrupts cancer

alphavbeta5, inhibition of

cell adhesion and invasion.

platelet aggregation, and

inhibition of cancer cell

adhesion to fibronectin and

vitronectin. (Arch

Biochem Biophys Mar. 15, 2000;

375(2): 278-288).

Corticotropin releasing

LocusID: 1393

WO9213074

Corticotropin-releasing factor (CRF) is a

The activity of CRF

Rheumatoid Arthritis;

factor binding protein

NP_001873

WO9410333

peripheral and a central mediator of

binding protein can be

Inflammation

XP_003672

US5733790

inflammation and stress-related

measured using a CRF

Genbank: P24387

responses. CRF-binding protein

binding assay (Peptides

Genbank: CAA41086

inactivates CRF.

Jan. 22, 2001; 22(1): 47-56)

CTLA4 (BMS 188667;

LocusID: 1493

WO9957266

CTLA4 is a B7 ligand receptor

CTLA4 Ig activity can be

Organ Transplant

BMS 224818; LEA29Y)

NP_005205

US5977318

normally expressed on activated T cells.

measured using a T cell

Rejection; Allergies;

XP_002490

US5968510

CTLA4 Ig is a soluble chimeric

activation assay (J

Rheumatoid Arhtritis;

US5844095

receptor with aintiinflammatory activity.

Immunol Mar. 1, 2001;

Graft-Versus-Host

US5885579

166(5): 3143-3150).

Disorders; Psoriasis;

Type 1 Diabetes

Mellitus; Xenotransplant

Rejection

Decorin

LocusID:

WO9956763-A1

This member of the specialized

Decorin function may be

Cancer; Diabetic

1634

WO9601842-A1

collagens and SLRP family is a small

measured using an in

Nephropathies;

XP_012239

WO9320202-A

proteoglycan that interacts with collagen

vitro collagen fibril

Inflammatory Disorders;

NP_001911

and growth factors, and is involved in

stability assay (Cell Mol

Post-Surgery Adhesions;

epithelial/mesenchymal interactions

Life Sci 2000 May;

Pulmonary Fibrosis

during organ development and shaping.

57(5): 859-863); or an in

vitro cell adhesion assay

(J Cell Biochem Oct. 1, 1997;

67(1): 75-83).

Del-1 (Developmentally-

LocusID: 10085

WO9515171

a new ligand for the alphavbeta3 integrin

Del-1 function may be

Ischemia; Cancer;

regulated endothelial

Genbank: U70312

WO9640769

receptor and may function to regulate

assayed in vitro using an

Restenosis

locus-1; EDIL3; EGF-

NP_005702

US5877281

vascular morphogenesis or remodeling

alphavbeta3 integrin

like repeats and

XP_003954

in embryonic development

adhesion assay. (Genes

discoidin I-like domains

Dev Jan. 1, 1998;

3)

12(1): 21-33).

Desmoteplase (Bat-tPA;

Genbank: AAA31591

EP383417-A

Tissue-type plasminogen activator;

Wallen, P., Biochemistry

Myocardial Infarction;

Desmodus salivary

Genbank: AAA31592

EP352119-A

serine protease that converts inactive

of plasminogen. In:

Thrombosis

plasminogen activator;

Genbank: P98119

plasminogen to plasmin

Kline D. L., Reddy,

DSPA alpha 1)

Genbank: JS0597

K. N. N., eds.

Fibrinolysis. Boca

Raton, FL: CRC Press,

1980: 1-25; Saksela, O.,

Rifkin, D. B., Cell-

associated plasminogen activation:

Regulation

and physiological functions.

Annu Rev Cell Biol 1988; 4:

93-126; Womack C J, Ivey

F M, Gardner A W,

Macko R F, Fibrinolytic

response to acute exercise

in patients with peripheral arterial

disease.

Med Sci Sports Exerc

2001 Feb; 33(2): 214-9

DNASE (Dornase alfa;

CAS-

WO9007572

Dnase is an endonuclease that

Dnase activity can be

Chronic Obstructive

Pulmozyme)

143831-71-4

WO9325670

selectively cleaves DNA and removes

measured using the DNA

Pulmonary Disease;

CAS-9003-

WO9410567

DNA from nuclear antigens at sites of

degradation assay

Cystic Fibrosis; Lupus

98-9

high cell turnover

described in J Biochem

Nephritis

CAS-

(Tokyo). 1982

132053-08-0

Oct; 92(4): 1297-303.

LocusID: 1773

NP_005214

XP_008097

Ectoapyrases (CD39

LocusID: 953

WO200004041-A2

Mediates catabolism of extracellular

Ectoapyrase activity may

Myocardial Infarction;

family; human

LocusID: 954

WO200023094-A2

nucleotides

be assayed in vitro using

Stroke

ectoapyrase (ecto-

LocusID: 955

WO200110205

an ectoapyrase assay. (J

ADPases); CD39-L2;

LocusID: 956

WO9630532-A1

Biol Chem Sep. 18, 1998;

CD39-L4)

LocusID: 957

WO9632471-A2

273(38): 24814-24821).

NP_001767

WO9946380-A2

NP_001237

NP_001238

NP_001239

NP_001240

XP_005712

XP_011771

XP_009435

XP_003296

XP_007435

EGF (Epidermal growth

CAS-62229-

WO9401553

EGF has bifunctional regulatory

Cell growth assay (J Biol

Gastric Ulcers; Wound

factor; EGF-genistein

50-9

WO9418227

properties: it inhibited the growth of

Chem Mar. 31, 1995;

Healing; Coronary

conjugate; EGF

LocusID: 1950

WO8500369

several epithelial tumor cells and

270(13): 7495-500)

Restenosis; Vascular

diphtheria toxin

NP_001954

US4966837

stimulated the growth of fibroblast and

Restenosis; Non-Small

chimeric protein)

XP_003608

other cell types

Cell Lung Cancer;

Psoriasis

EMAP II (Endothelial

LocusID: 9255

WO9710841-A1

EMAP-II is a tumor-derived cytokine

EMAP II activity may be

Cancer

monocyte activating

LocusID: 13722

WO9808950-A1

which inhibits angiogenesis, exhibits

assayed in vitro using a

polypeptide II)

NP_004748

WO9640719-A1

protperties of a proinflammatory

capillary sprouting assay

XP_003390

US6013483-A

mediator, and exerts potent effects on

(Curr Biol Apr. 23, 1998;

endothelial cells in vitro and in vivo.

8(9): 529-532).

FGF-1

LocusID: 2246

EP298723-A

FGF-1 stimulates proliferation and

Cell proliferation assay:

Peripheral Vascular

NP_000791

US5571790-A

angiogenesis

Cell, vol.50, no.5,

Disease; Peripheral

US5827826-A

pp.729-737 (Aug. 1987).

Arterial Occlusive

US5849538-A

Proc Natl Acad Sci U.S.A,

Disease; Critical Limb

US6037329-A

vol.86, no.3, pp.802-806

Ischemia

AUS6077692-A

(1989).

US6077692-A

WO8705332-A

WO9503831-A

WO9524928-A2

FGF-2 (Fibroblast

CAS-

EP237966-A

Fibroblast growth factor. Angiogenic

Proliferation assay using

Coronary Disorders;

Growth Factor-2;

131094-16-1

EP226181-A

growth factor

NR6R-3T3 cells

Fracture; Periodontal

Fiblast)

LocusID: 2247

WO9005184-A

(Rizzino 1988 Cancer

Disease; Peripheral

NP_001997

WO9202539-A

Res. 48: 4266)

Vascular Disease;

XP_003306

WO9325688-A

Postmenopausal

US5604293-A

Osteoporosis; Skin Ulcer;

US6083706-A

Stroke; Peripheral Artery

US6037329-A

Disease; Coronary Artery

Disease

Fibrolase (Zinc-

CAS-

EP323722-A

Fibrolase is a zinc metalloproteinase

Fibrolase activity may be

Thrombosis; Stroke

containing

116036-70-5

WO9846771-A2

possessing direct-acting fibrinolytic

assayed in vitro using a

metalloproteinase;

Genbank: AAB26922

activity purified from southern

fibrinolytic assay.

(Southern copperhead

copperhead (Agkistrodon contortrix

(Thromb Res May 15, 1994;

snake) Agkistrodon

contortix) snake venom. It cleaves the A

74(4): 355-367).

contortrix contortrix)

alpha-chain of fibrinogen initially at a

single site: Lys413-Leu.

FLT3 ligand (Mobista)

LocusID: 2323

WO9426891-A

Stimulates growth and survival of

Proliferation assay using

Chemoprotection;

NP_001450

EP627487-A

hematopoitic progenitor cells.Stimulates

a Flt-3 transformed pro

Haematological

XP_008921

WO9857655-A1

dendritic cell development.

B-cell line (Hannum

Disorders; Malignant

WO9818923-A1

1994 Nature 368: 643)

Melanoma;

Myelosuppression; Non-

Hodgkin's Lymphoma;

Prostate Cancer; Stem Cell

Mobilization

Follitropin (Follitropin

CAS-

WO8810270

Follitropin is a pituitary glycoprotein

Follitropin activity may

Female Infertility; Male

alpha; Follitropin beta;

146479-72-3

EP735139

hormone which stimulates

be assayed in vitro by

Infertility

Follicle Stimulating

CAS-56832-

US6083706

steroidogenesis via a cAMP pathway.

measuring cAMP

Hormone;

30-5

WO8604589

production in cells

Urofollitropin; ORG

CAS-

US5177193

expressing the FSH

32489; Gonal-F;

110909-60-9

US5639639

receptor. (J Reprod

Follistim; Puregon;

CAS-

US5712122

Immunol 2001 Jan;

FERTINEX; Metrodin

150490-84-9

US5767251

49(1): 1-19).

HP)

CAS-97048-

13-0

LocusID: 1081

LocusID: 2488

NP_000726

NP_000501

XP_011444

XP_006316

GDNF (Glial-derived

CAS-

WO9846737-A2

Glial-derived neurotrophic factor

GDNF activity may be

Amyotrophic Lateral

neurotrophic factor;

185857-51-6

DE19816186-A1

(GDNF) is a distant member of the

assayed in vitro by

Sclerosis; Parkinson's

Neurturin)

LocusID: 2668

WO9719693-A1

transforming growth factor-beta (TGF-

measuring increases in

Disease; Huntingdon's

NP_000505

WO9730722-A1

beta) superfamily which acts

Ret tyrosine

Disease

XP_003703

intracellularly via the receptor tyrosine

phosphorylation in

kinase Ret. GDNF is a potent survival

response to GDNF

factor for midbrain dopamine neurons,

treatment. (Mol Cell Biol

motoneurons, noradrenergic neurons, as

Mar. 15, 1995; (3): 1613-1619).

well as for sympathetic, parasympathetic and sensory

neurons. However, for most neuronal populations,

except for motoneurons, TGF-beta is required as a

cofactor for GDNF. GDNF also has

distinct functions outside the nervous system,

promoting ureteric branching in

kidney development and regulating spermatogenesis.

Gelsolin

LocusID: 2934

WO9518221

Gelsolin is a calcium-dependent protein

Gelsolin activity may be

Bronchitis; Cystic

NP_000168

WO9852597

which severs and caps actin filaments,

assayed in vitro by

Fibrosis

Genbank: CAA28000

and has been shown to decrease the

measuring proteolysis of

viscosity of cystic fibrosis sputum.

actin. (Nature 1987 Jan

22-28; 325(6102): 362-364).

Glial growth factor-2

LocusID: 3084

WO9630403-A1

GGF2 is an isoform of neuregulin 1,

GGF2 activity may be

Multiple Sclerosis;

(GGF-2; Neuregulin 1)

NP_039256

WO9630403-A1

containing a kringle-like sequence plus

assayed in vitro by

Chemotherapy-Induced

Genbank: AAB59622

WO9426298-A

Ig and EGF-like domains. The GGF2

measuring activation of

Neuropathy; AIDS

WO9400140-A

receptor is a member of the ERBB

ERBB receptor tyrosine

Neuropathy; Diabetic

WO9218627-A

family of tyrosine kinase transmembrane

kinases in human

Neuropathy; Peripheral

receptors. Through interaction with

rhabdomyosarcoma cells.

Neuropathies

ERBB receptors, GGF2 plays a central

(Int J Cancer Jul. 1, 2000;

role in neuronal and glial development

87(1): 29-36).

in the central nervous system.

Glucagon (Glucagon;

CAS-16941-

WO9726321-A2

Glucagon is a 29 amino acid peptide

Glucagon's glucogenic

Hypoglycemia;

Insulinotropin;

32-5

US5958909-A

hormone liberated in the A cells of the

activity is mediated by a

Gastrointestinal Tract

GLUCAGON;

CAS-

US6110707-A

islets of Langerhans. Glucagon is

high affinity glucagon

Diagnostic; Diabetes

GLUCAGEN)

118549-37-4

EP612531-A

produced in response to a drop in blood

receptor. Biological

Mellitus; Obesity; Type

LocusID: 2641

sugar concentration. Its effect is to raise

acitivity of recombinant

2 Diabetes Mellitus

NP_002045

serum glucose. In the treatment of severe

glucagon can be assesed

XP_002210

hypoglycaemia in insulin-dependent

by direct binding assays

diabetics, glucagon causes the liver to

(Science Mar. 12, 1993;

release glucose by stimulating the

259(5101): 1614-6).

conversion of glycogen to glucose.

Glucagon also relaxes gastrointestinal smooth

muscle, allowing for improved radiological exams.

HBNF (Heparin-binding

LocusID: 5764

EP535337-A

HBNF stimulates neurite outgrowth in

HBNF activity may be

Neuropathies;

neurotrophic factor;

NP_002816

EP569703-A

neurons and is expressed in a

assayed in vitro using a

Neurological Disorders

PTN; pleiotrophin;

Genbank:

EP441763-A

developmentally regulated manner in the

neurite outgrowth assay.

heparin binding growth

AAA41311

EP474979-A

rat brain.

(J Biol Chem Oct. 27, 1995;

factor 8; neurite growth-

Genbank:

WO200035473-A2

270(43): 25992-25999).

promoting factor 1;

M68916

NEGF1)

HCG (Human chorionic

LocusID: 1081

WO8810270

Human chorionic gonadotropin is a

hCG receptor binding and

Breast Cancer; Kaposi's

gonadotropin; Pregnancy

LocusID: 1082

EP735139

heterodimer of a common alpha chain

activation can be

Sarcoma; Female

Urine Hormone;

NP_000726

US6083706

and a unique beta chain (chorionic

measured to assess

Infertility;

Ovidrel; Ovidrelle;

NP_000728

WO9749418-A1

gonadotropin beta), which confers

biological activity of

Hypogonadism; Male

APL)

XP_011444

WO9749721-A1

biological specificity to a number of

recombinant hCG (J Biol

Infertility

XP_012903

WO9522340-A1

glycopeptide hormones including

Chem Oct. 5, 1993;

US5864488-A

thyrotropin, lutropin, follitropin and

268(28): 20851-4).

gonadotropin. Only the heterodimer is

functional. Its normal function is to

stimulate the ovaries to synthesize the steroids

that are essential for the maintenance of pregnancy.

Heat shock protein

LocusID: 3316

US6168793

Heat shock proteins chaperone antigenic

Administration of HSP-

Solid Tumors; Colorectal

(HSP; HSP96; gp96;

Genbank: M11717

US6136315

peptides in the display pathway. Gp96

peptide complexes in two

Cancer; Gastric Cancer;

ONCOPHAGE)

Genbank: M15432

US5958416

elicits a tumor-specific killing response

UV-induced carcinoma

Malignant Melanoma;

Genbank: X15183

US5837251

when complexed with antigenic tumor

models in mice (US

Non-Hodgkin's

Genbank: M33716

antigen.

5,837,251)

Lymphoma; Pancreatic

Cancer; Renal Cancer;

Sarcoma; Epilepsy;

Neuroprotection; Stroke

IL-1 (Interleukin-1;

LocusID: 3552

WO9728808

The cytokine interleukin-1 (IL-1)1

1) binding to IL-1

Bacterial Infections;

interleukin-1 alpha;

LocusID: 3553

EP324447

elicits a wide array of biological

receptors in YT-NCI or

Peptic Ulcer; Transplant

interleukin-1 beta)

NP_000566

US6083706

activities that initiate and promote the

C3H/HeJ cells (Carter et

Rejection; Cancer;

NP_000567

host response to injury or infection,

al., Nature 344: 633-638,

Malignant Melanoma;

XP_010760

including fever, sleep, loss of appetite,

1990); 2) induction of

Non-Small Cell Lung

acute phase protein synthesis,

endothelial cell-leukocyte

Cancer; Preleukemia;

chemokine production, adhesion

adhesion (Carter et al.,

Chemoprotection;

molecule up-regulation, vasodilatation,

Nature 344: 633-638,

Radioprotection

the pro-coagulant state, increased

1990); 3) proliferation

hematopoiesis, and production and

assays on A375-C6 cells

release of matrix metalloproteinases and

(Murai T et al., J. Biol.

growth factors.

Chem. 276: 6797-6806,

2001); D10S

proliferation: Orencole &

Dinarello (1989) Cytokine 1, 14-20

IL-1 receptor antagonist

CAS-

WO9117249-A

The IL1 receptor antagonist is a protein

1) competition for IL-1

Rheumatoid Arthritis;

(Anakinra; soluble

143090-92-0

US5817306-A

that binds avidly to IL1 receptors

binding to IL-1 receptors

Asthma; Diabetes

interleukin-1 receptor;

LocusID: 3557

US5747444-A

without activating the target cells and

in YT-NCI or C3H/HeJ

Mellitus; GVHD;

IRAP; KINERET;

NP_000568

US5932537-A

inhibits the binding of IL1-alpha and

cells (Carter et al., Nature

Inflammatory Bowel

ANTRIL)

XP_010756

IL1-beta. As a consequence, the biologic

344: 633-638, 1990); 2)

Disorders; Ocular

activity of these 2 cytokines is

inhbition of IL-1-induced

Infammation; Psoriasis;

neutralized in physiologic and

endothelial cell-leukocyte

Septic Shock; Transplant

pathophysiologic immune and

adhesion (Carter et al.,

Rejection; Inflammatory

inflammatory responses.

Nature 344: 633-638,

Disorders; Rheumatic

1990); 3) proliferation

Disorders;

assays on A375-C6 cells,

Postmenopausal

a human melanoma cell

Osteoporosis; Stroke

line highly susceptible to

the antiproliferative action

of IL-1 (Murai T et al., J.

Biol. Chem. 276: 6797-6806,

2001).

IL-10 (Cytokine

LocusID: 3586

WO9212725-A

IL-10 inhibits the synthesis of a

1) binding of IL-10 to

Inflammatory Bowel

synthesis inhibitory

NP_000563

WO9302693-A

number of cytokines, including ifn-

NK cells (carson WE et

Diseases; Acute Lung

factor; rhIL-10; SCH

XP_001409

US5231012-A

gamma, il-2, il-3, tnf and gm-csf

al., Blood 85: 3577-3585,

Injury; Autoimmune

52000; TENOVIL)

US5837293-A

produced by activated macrophages and

1995); 2)

Disorders; Cancer;

US5833976-A

by helper t cells.

inhibition of TNF-alpha

Crohn's Disease; Graft-

US6106823-A

production by

Versus-Host Disorders;

macrophages (Riley J K et

Growth Disorders;

al., J. Biol. Chem. 274:

Hepatic Fibrosis;

16513-16521, 1999); 3)

Hepatitis C; Herpes

inhibition of macrophage

Simplex Virus Infections;

proliferation (O'Farrell A-

HIV Infections

M et al., EMBO 17:

Treatment;

1006-1018, 1998); MC-9

Ischaemia/Reperfusion;

proliferation: Thompson-

Multiple Sclerosis;

Snipes et al (1991) J.

Myocarditis; Psoriasis;

Exp. Med. 173, 507-510

Rheumatoid Arthritis;

Sepsis; Transplant

Rejection; Type 1

Diabetes Mellitus;

Ulcerative Colitis;

Rheumatoid Arthritis;

Inflammatory Bowel

Diseases

IL-11 (Oprelvekin;

CAS-

WO9107495-A

stromal cell-derived cytokine, initially

hematopoietic cell

Thrombocytopenia; Bone

recombinant human

145941-26-0

US6054294-A

characterized as a hematopoietic

proliferation assay.

Marrow Transplant

interleukin-11; SCH

LocusID: 3589

WO9405318-A

cytokine with thrombopoietic

“Interleukin-11 enhances

Rejection; Crohn's

53620; YM 294;

NP_000632

WO9513393-A

activity, IL-11 has now been shown to

human

Disease; Graft Versus

NEUMEGA)

XP_008906

be expressed and exhibits pleiotropic

megakaryocytopoiesis in

Host Disorders;

action on hematopoietic cells and

vitro.”

Inflammatory Disorders;

multiple other tissues, including brain,

Blood. Jan. 15, 1992;

Mucositis; Rheumatoid

spinal cord neurons, gut, and testis.

79(2): 327-31.

Arthritis; Sepsis-Induced

“Synergistic effects of

Systemic Inflammatory

stem cell factor and

Response Syndrome

interleukin 6 or

interleukin 11 on the expansion

of murine

hematopoietic progenitors

in liquid suspension

culture.” Stem Cells.

1995 Jul; 13(4): 404-13;

B9-11 proliferation: Lu et

al (1994) J immunol.

Methods 173, 19

IL-12 (Interleukin-12;

LocusID: 3592

US5571515-A

IL-12 exhibits antitumor, antiviral, and

natural killer (NK) cell

Cancer; Hepatitis C;

CLMF; Edodekin alfa;

LocusID: 3593

US5723127-A

antimicrobial activities and can modify

cytotoxicity assay and

Asthma; Bacterial

IL-12; Interleukin 12;

NP_000873

US5756085-A

allergic inflammatory reactions in

interferon-gamma (IFN-

Infections; Cancer;

NKSF; Recombinant

NP_002178

US5780597-A

animal models. Animals and humans

gamma) release assay.

Cutaneous T-Cell

human interleukin-12;

XP_003121

US5976539-A

genetically deficient in IL-12 are highly

“Requirement for type 2

Lymphoma; Graft-Versus-

rhIL-12; rHuIL-12; RO

XP_004011

US5994104-A

susceptible to mycobacteria and

NO synthase for IL-12

Host Disorders; Hepatitis

247472; YM 01C)

WO9205256-A

salmonella. IL-12 is principally a

signaling in innate

B; HIV Infections

WO9519786-A1

phagocyte-derived cytokine that targets

immunity”. Science 284:

Treatment; Kaposi's

natural killer cells and T lymphocytes,

951-955, 1999; KIT—225

Sarcoma; Leishmaniasis;

stimulating their activity and the

proliferation: Hori et al

Lymphoma; Malaria;

secretion of interferon gamma.

(1987), Blood 70, 1069-1078

Malignant Melanoma;

Non-Hodgkin's

Lymphoma; Renal Cancer

IL18 binding protein

LocusID: 10068

WO9909063-A1

IL18 binding protein inhibits the early

IL18 binding protein

Autoimmune Disease;

(Interleukin 18 binding

NP_005690

WO200012555-A1

Th1 cytokine response, it is a member

activity may be assayed

Inflammation;

protein)

XP_006006

of the immunoglobulin superfamily.

in vitro by measuring its

Rheumatoid Arthritis

ability to inhibit the early

Th1 response. (Immunity

1999 Jan; 10(1): 127-136).

IL2-diphtheria toxin

LocusID: 3558

WO8505124-A

Binds to and kills cells expressing the

IL2-diphtheria toxin

Diabetes Mellitus; HIV

chimera (Interleukin-2

NP_000577

EP215576-A

IL2 receptor.

chimera function may be

Infection; Rheumatoid

diphtheria toxin

NM_000586

EP147819-A

assayed in vitro using a

Arthritis; Cancer

chimeric protein)

K01722

cytotoxicity assay. (Exp

AAA32182

Hematol 2000 Dec;

28(12): 1390-1400).

IL-3 & G-CSF fusion

CAS-

WO8905824

Myelopoietins (MPOs) are a family of

CFU-GM colonies in a

Myelosuppression; Stem

protein (Leridistim; SC

193700-51-5

WO8806161

engineered dual interleukin-3 (IL-3) and

human bone marrow-

Cell Mobilization

68420; myelopoietin)

LocusID: 3562

WO8800598

granulocyte colony-stimulating factor

derived CD34+ colony-

NP_000579

(G-CSF) receptor agonists that are

forming unit assay.

XP_003752

superior in comparison to the single

(PMID: 10194378)

agonists in their ability to promote the growth and

maturation of hematopoietic cells of the myeloid lineage.

IL-4 (Interleukin-4;

LocusID: 3565

US5986059-A

A fusion protein comprised of IL-4 and

Cytotoxicity assay in

Cancer; Acute

BSF-1; SCH 39400;

NP_000580

WO9747744-A2

truncated Pseudomonas exotoxin has

normal T lymphocytes.

Lymphoblastic

interleukin-4-

XP_004053

WO9803654-A2

anti-tumor activity for tumors

(PMID: 8144944);

Leukaemia;

Pseudomonas exotoxin;

Genbank: P11439

US6028176-A

expressing IL-4 receptor. This fusion

RAMOS Augmentation

Gastrointestinal Cancer;

NBI 3001;

protein is in clinical trial for treating

of CD23 expression:

Immunodeficiency

QUADRAKINE)

breast cancer, glioma, glioblastoma,

Siegel & Mostowski

Disorders; Inflammation;

refractory hairy cell leukemia, and

(1990) J Immunol

Kaposi's Sarcoma;

AIDS-associated Kaposi's sarcoma

Methods 132, 287-295

Malignant Melanoma;

tumors.

Non-Small Cell Lung

Cancer; Rheumatoid

Arthritis; Solid Tumours;

Type 1 Diabetes

Mellitus; Breast Cancer;

Colorectal Cancer; Gastric

Cancer; Glioma; Kaposi's

Sarcoma; Lymphoma;

Malignant Melanoma;

Prostate Cancer; Renal

Cancer;

IL-4 Receptor

LocusID: 3566

EP367566

Binds to and can block activity of IL4

Activity can be measured

Asthma; Graft-Versus-

(interleukin-4 receptor;

NP_000409

US5599905

which has a wide range of effects

by ability to inhibit IL-4

Host Disorders;

NUVANCE)

XP_007989

US5767065

including inducing B-cells to proliferate

dependent proliferation of

Hypersensitivity

US5717072

and induce cytokine production;

TF-1 cells (Kitamura, T

US5856296

inducing maturation of thymocytes;

et al., 1989, J. Cell.

US5840869

activation of mature T-cells, NK cells,

PPhysiol. 140:323)

modulates myelopoesis, enhancement of

eosinophil and mast cell genertation;

prolifertaion of endothelial cells.

IL4-diphtheria toxin

LocusID: 3565

US5986059-A

Binds to and kills cells expressing the

IL4-diphtheria toxin

Autoimmune Disease;

chimera (Interleukin-4

NP_000580

WO9747744-A2

IL4 receptor.

chimera function may be

HIV Infection; Transplant

diphtheria toxin

K01722

WO9803654-A2

assayed in vitro using a

Rejection; Cancer;

chimeric protein;

AAA32182

US6028176-A

cytotoxicity assay. (Exp

Hypersensitivity;

DAB389 interleukin-4;

Hematol 2000 Dec;

Leukemia; Lymphoma

Interleukin-4 fusion

28(12): 1390-1400).

toxin)

IL6-diphtheria toxin

LocusID: 3569

EP220574-A

Binds to and kills cells expressing the

IL6-diphtheria toxin

HIV Infection; Cancer

chimera (Interleukin-6

NP_000591

WO8800206-A

IL6 receptor.

chimera function may be

diphtheria toxin

K01722

EP326120-A

assayed in vitro using a

chimeric protein;

AAA32182

WO8905824-A

cytotoxicity assay. (Exp

DAB389 IL-6)

US6054294-A

Hematol 2000 Dec;

28(12): 1390-1400).

IL6-Pseudomonas

LocusID: 3569

EP220574-A

Binds to and kills cells expressing the

IL6-pseudomonas

Cancer; Myelocytic

exotoxin chimera

NP_000591

WO8800206-A

IL6 receptor.

exotoxin chimera function

Leukemia

(Interleukin-6

Genbank: P11439

EP326120-A

may be assayed in vitro

Pseudomonas exotoxin

WO8905824-A

using a cytotoxicity

chimeric protein; IL-

US6054294-A

assay. (Proc Natl Acad

6PE4E)

Sci USA 1989 Jun;

86(11): 4215-4219).

IL-8 (Interleukin-8)

LocusID: 3576

WO8910962-A

IL-8 Interleukin 8 is a cytokine that

Calcium reflux in IL8R

Labor Induction

NP_000575

WO9000563-A

plays a role in chemoattraction and

bearing cells: Holmes et

XP_003501

US5871723-A

activation of neutrophils. It has

al (1991)Science 253,

similarity to several platelet-derived

1278-80

factors.

Interferon gamma

CAS-98059-

EP343388-A

Anti-vial, anti-viral and

Can measure activity in

Serious Infections

(Interferon gamma 1b;

61-1

US4835256-A

immunomodulatory activities.

anti-viral assay using

Associated With Chronic

SUN 4800; OH 6000;

LocusID: 3458

WO8502624-A

Hela cells infected with

Granulomatous Disease;

IMMUKIN;

NP_000610

WO8504186-A

EMC virus: Meager, A.

Severe; Malignant

ACTIMMUNE;

XP_006883

1987, Lymphokines and

Osteopetrosis;

BIOGAMMA;

Interferons, A Practical

Tuberculosis; Atopic

OGAMMA)

Approach, Clemens, MJ

Dermatitis; Chronic

et al., eds., IRL Press, p.

Granulomatous Disease;

129.Can measure

Cryptococcoses; Cystic

modulation of MHC class

Fibrosis; Keloids;

II expression on Human

Malignant Melanoma;

colorectal carcinoma cell

Mycobacterial Infections;

line COLO 205: Gibson

Mycoses; Ovarian

and Kramer, 1989, J.

Cancer; Pulmonary

Immunol. Methods,

Fibrosis; Renal Cancer;

125: 105-113

Small Cell Lung Cancer;

Systemic Scleroderma;

Mycosis Fungoides; Skin

Cancer

Interferon omega (IFN

LocusID:

EP174143-A

Regulates antiviral defence, cell growth,

Interferon-omega activity

Cancer; Hepatits C

omega; Biomed 510)

3467

EP170204-A

and immune activation; member of the

can be measured using an

NP_002168

WO9806431-A2

type I interferon family of proteins.

in vitro antiviral assay (J

XP_005411

EP236920-A

Med Microbiol 1998

Nov; 47(11): 1015-8).

Interferon-inducible

LocusID:

WO9504158-A

IFN-gamma-inducible protein is a

rIP-10 activity may be

Leukaemia

protein 10 (rIP-10)

3627

WO9700691-A1

member of the C-X-C chemokine

assayed in vitro using a

NP_001556

US5871723-A

family, and has chemotactic and

rat artery smooth muscle

CAA26370

mitogenic activity.

cell chemotaxis assay. (J

Biol Chem Sep. 27, 1996;

271(39): 24286-24293).

Interleukin-3 (IL-3; SDZ

CAS-

WO8905824-A

Interleukin-3 (colony-stimulating factor)

IL-3 activity may be

Cancer; Hematological

215134,

148641-02-5

WO8806161-A

is a member of a family of growth factors

measured in vitro using a

Disorders; Hematological

ALLEVORIN;

CAS-

WO8800598-A

which plays a role in hematopoeisis. It

haematopoietic cell

Malignancies;

Hemokine; ILE 964;

161753-30-6

is capable of supporting the of a broad

differentiation assay.

Preleukemia; Hodgkin's

Muplestim; SDZ ILE

LocusID:

range of hematopoietic cell types.

(Science 1985;

Disease; Myeloid

964; SDZ ILE964;

3562

228(4701): 810-815).

Leukemia; Non-

synthokine; SC 55494)

NP_000579

Hodgkin's Lymphoma;

XP_003752

Thrombocytopenia;

Chemoprotection;

Radioprotection

KGF-1 (Keratinocyte

LocusID: 2252

WO9524928-A2

Keratinocyte growth factor 1 (KGF-1) is

KGF-1 activity may be

Chemoprotection; Graft-

growth factor-1; AMJ

NP_002000

US6077692-A

a human mitogen that is specific for

assayed in vitro using an

Versus-Host Disorders;

9701; FGF-7; Fibroblast

US6037329-A

epithelial cells. It has the properties of a

epithelial cell

Mucositis

growth factor 7)

WO9008771-A

stromal mediator of epithelial cell

proliferation assay. (Proc

WO9611950-A1

proliferation.

Natl Acad Sci U.S.A. 1989

WO9501434-A

Feb; 86(3): 802-806).

EP619370-A

WO9611952-A1

Kistrin (Malayan pit

Genbank: P17

WO9015072

Kistrin is a 68-amino acid polypeptide

Kistrin activity may be

Thrombosis; Stroke;

viper)

494

US5686566

from the venom of the Malayan pit viper

assayed in vitro by

Ischemic Heart Disorders

US5686568

(Agkistrodon rhodostoma), which

assaying thrombolysis,

US5686570

inhibits the platelet GPIIb/IIIa receptor

reocclusion, and bleeding

which results in effects on thrombolysis,

associated with

reocclusion, and bleeding.

administration of

recombinant tissue-type

plasminogen activator (rt-PA)

in a canine model of

coronary artery

thrombosis. (Circulation

1991 Mar; 83(3): 1038-1047).

Kunitz protease inhibitor

LocusID:

PCT/US00/31917

Kunitz protease inhibitor 1 is a potent

Kunitz protease inhibitor

Thrombosis;

1 (KPI 1)

6692

inhibitor specific for HGF activator and

1 activity may be assayed

Anticoagulant

NP_003701

is thought to be involved in regulation

in vitro by measuring

XP_007555

of proteolytic activation of HGF in

inhibitory activity toward

injured tissues.

HGF activator. (J Biol

Chem Mar. 7, 1997;

272(10): 6370-6376).

Lactoferrin

Locus

WO9325567-A

Lactoferrin, a member of the transferrin

Lactotransferrin activity

Allergic Contact

(Apolactoferrin; GPX

ID: 4057

WO9108216-A

family, transports iron in extracellular

can be measured using an

Dermatitis; Bacterial

400)

NP_002334

WO9013642-A

fluid, and has antibacterial and antiviral

in vitro viral inhibition

Infections; Cardiovascular

XP_010963

properties.

assay (J Med Microbiol

Disorders; Coagulation

1998 Nov; 47(11): 1015-8),

Disorders; Dry Eye;

and antimicrobial

Gastritis; Hepatitis C;

assays (J Clin Invest

Psoriasis

Sep. 1, 1998; 102(5): 874-80).

Leptin

LocusID: 3952

EP741187-A2

Secreted by fat cells. Obesity control.

in vivo modulation of

Obesity; Type 2 Diabetes

NP_000221

WO9622308-A2

Modulation of food intake, reduction of

food intake, reduction in

Mellitus; Vascular

XP_004625

WO9739767-A1

body weight, and lowering of insulin

body weight, and

Disorders; Immunological

US6124448-A

and glucose level.

lowering of insulin and

Disorders;

US6124439-A

glucose levels in ob/ob

Immunosuppression

WO9634111-A1

mice, radioimmunoassay

(RIA) and activation of

the leptin receptor in a

cell-based assay. Protein

Expr Purif 1998

Dec; 14(3): 335-42

Leukemia inhibitory

LocusID:

US6054294-A

Leukaemia inhibitory factor is a

LIF activity may be

Female Infertility;

factor (Emifilermin; LIF;

3976

WO9305169-A

cytokine that induces macrophage

measured in vitro using a

Neuromuscular Disorders

AM 424; Cholinergic

NP_002300

WO9418236-A

differentiation. LIF acts in conjunction

haematopoietic cell

differentiation factor)

XP_009915

WO9008188-A

with BMP2 on primary fetal neural

differentiation assay.

progenitor cells to induce astrocytes.

(Science 1985;

228(4701): 810-815).

LFA-3/IgG1 fusion

LocusID: 965

WO8809820-A

LFA-3/IgG1 is chimera of IgG1 with

LFA-3/IgG1 activity may

Autoimmune Disorders;

protein (AMEVIVE)

LocusID: 3500

EP517174-A

LFA-3 which interacts with the CD2

be assayed in vitro by

Inflammation; Psoriasis;

NP_001770

WO9012099-A

receptor and aids contact of helper T

measuring its ability to

Transplant Rejection;

XP_001325

WO9002181-A

cells with antigen presenting cells.

inhibit T-cell function. (J

WO9201049-A

Specific interaction of LFA-3 with CD2

Exp Med Jul. 1, 1993;

US5506126-A

can inhibit T-cell responses.

178(1): 211-222).

Lys plasminogen

Locus

WO9013640-A

A more reactive, truncated form of

Lys plasminogen activity

Thrombosis; Arterial

(Recombinant truncated

ID: 5340

US5200340-A

plasminogen. Plasminogen is the

can be measured using an

Occlusive Disorders

plasminogen)

NP_000292

WO9108297-A

precursor of plasmin, a serine protease

in vitro fibrinolysis assay

XP_004371

US5648254-A

that digests fibrin in clots.

(J Biol Chem Dec. 25, 1992;

267(36): 26150-6).

Maspin (LXR 023;

CAS-

WO9405804-A

A serine protease inhibitor that

Maspin activity can be

Cancer; Breast Cancer;

Maspin serine protease

157857-21-1

US5470970-A

suppresses tumor metastasis by

measured using in vitro

Prostate Cancer

inhibitor)

Locus

US5801001-A

inhibiting angiogenesis.

angiogenesis assays (Nat

ID: 5268

US5905023-A

Med 2000 Feb; 6(2): 196-9).

NP_002630

XP_008742

Methioninase (AC 9501;

CAS-42616-

WO9517908-A1

Methioninase catalyzes alpha, gamma-

Methioninase activity

Cancer; Gastrointestinal

AC 9301; Recombinant

25-1

US5888506-A

elimination reactions of homocysteine

may be assayed in vitro

Cancer; Non-Small Cell

methioninase; rMETase;

Genbank: AAB03240

US5891704-A

and its S-substituted derivatives as well

as described by Ito et al.

Lung Cancer; Pancreatic

ONCase)

WO9640284-A1

as alpha, beta-elimination reactions of

(J Biochem (Tokyo)

Cancer

cysteine and its derivatives. It also

1975 Nov; 78(5): 1105-1107).

catalyzes exchange reactions between the

substituent at the gamma-carbon of

homocysteine substrates and

exogenously added alkanethiols,

forming the corresponding S-alkylhomocysteines. It

also catalyzes similar beta-exchange reactions between

cysteine and alkanethiols.

Microsomal transfer

Locus

AU9334064-A

MTP encodes the large subunit of the

MTP activity may be

Lipid Disorders

protein (BMS 212122)

ID: 4547

heterodimeric microsomal triglyceride

assayed in vitro by using

NP_000244

transfer protein. Protein disulfide

an apoB-containing

XP_003363

isomerase (PDI) completes the

lipoprotein secretion

heterodimeric microsomal triglyceride

assay. (J Biol Chem

transfer protein, which has been shown

Sep. 2, 1994;

to play a central role in lipoprotein

269(35): 21951-21954).

assembly.

MSH-diphtheria toxin

Locus

WO200037615-A1

Binds to and kills cells expressing the

MSH-diphtheria toxin

Cancer

chimera (Melanocyte

ID: 5443

EP926239-A2

MSH receptor.

chimera function may be

Stimulating Hormone-

NP_000930

assayed in vitro using a

diphtheria toxin

XP_002485

cytotoxicity assay. (Exp

chimeric protein;

K01722

Hematol 2000 Dec;

DAB389 MSH; MSH

AAA32182

28(12): 1390-1400).

fusion toxin)

Nerve growth factor

LocusID:

EP121338-A

A growth factor with roles in neuronal

NGF activity may be

HIV-Associated Sensory

(NGF)

4803

EP414151-A

differentiation and survival.

assayed by measurement

Neuropathy; Diabetic

NP_002497

WO9530686-A1

of CREB transcription

Neuropathy;

XP_002122

WO9310150-A

factor activation in

Neurodegenerative

sympathetic neurons in

Disorders

culture. (Science Dec. 17, 1999;

286(5448): 2358-2361).

Neutral endopeptidase

CAS-82707-

EP596355-A

Neutral endopeptidase is a 100-kD type

Neutral endopeptidase

Cancer; Migraine;

(NEP; rNEP)

54-8

EP272928-A

II transmembrane glycoprotein that is

activity may be assayed

Inflammatory Bowel

Locus

WO8905353-A

particularly abundant in kidney, where it

in vitro by measuring

Disease; Inflammation;

ID: 4311

EP596355-A

is present on the brush border of

proteolysis of bombesin-

Asthma; Respiratory

NP_000893

US5736376-A

proximal tubules and on glomerular

like peptides. (Proc Natl

Disease; Lung Cancer;

NP_009218

US5688640-A

epithelium. NEP cleaves peptides at the

Acad Sci U.S.A. Dec. 1, 1991;

Small Cell Lung Cancer

NP_009219

amino side of hydrophobic residues and

88(23): 10662-10666).

NP_009220

inactivates several peptide hormones

XP_003136

including glucagon, enkephalins,

XP_003137

substance P, neurotensin, oxytocin, and

XP_003138

bradykinin.

XP_003139

NIF (Neutrophil

Genbank: AAA27789

WO9323063-A

NIF is a glycoprotein inhibitor of a

NIF activity may be

Stroke

inhibitory factor;

US5919900-A

number of neutrophil functions, such as

assayed in vitro using a

Corleukin; UK 279276)

adhesion to endothelial cells and

polymorphonuclear

adhesion-dependent release of hydrogen

leukocyte adhesion assay.

peroxide. These functional effects

(Mol Pharm 1999 Nov;

resulted from its specific binding to

56(5): 926-932).

alphaMbeta2 but not to other beta2

integrins. NIF has been shown effective

in attenuating the deleterious effects of excessive

neutrophil activation, such as tissue damage

and ischemia-reperfusion injury in animal models

Noggin

Locus

WO9903996-A1

Noggin binds and inactivates members

Noggin activity may be

Fibrodysplasia Ossificans

ID: 9241

WO9405791-A

of the transforming growth factor-beta

assayed by measuring

Progressiva; Abnormal

NP_005441

(TGF-beta) superfamily signaling

TNF receptor responses

Bone Growth;

XP_008151

proteins, such as bone morphogenetic

to TNF stimulation of

Pathological Bone

protein-4 (BMP4).

HeLa cells. (J Biol Chem

Growth

Feb. 28, 1997;

272(9): 5861-5870).

NT-3 (Neurotrophin 3)

LocusID: 4908

WO9103569

NT-3 is a member of a family of

NT-3 activity may be

Enteric Neuropathies;

NP_002518

WO9310150

neurotrophic factors, neurotrophins, that

assayed in vitro by

Constipation; Diabetic

WO9530434

control survival and differentiation of

measuring its ability to

Neuropathies; Peripheral

mammalian neurons. This gene is

proliferate cultured NC

Nerve Disorders

closely related to both nerve growth

progenitor cells grown in

factor and brain-derived neurotrophic

a serum-free defined

factor. The protein encoded by this gene

medium. (Proc Natl Acad

may be involved in the maintenance of

Sci U.S.A Mar. 1, 1992;

the adult nervous system, and affect

89(5): 1661-1665).

development of neurons in the embryo when it is

expressed in human placenta.

Osteogenic protein-1

LocusID: 655

WO9011366-A

OP-1 is a member of the transforming

OP-1 activity may be

Acute Fractures; Cartilage

(OP-1; BMP-7; Bone-

NP_001710

WO9105802-A

growth factor-beta superfamily of

assayed in vitro by

Repair; Neurological

related protein; OP-1

XP_012943

WO9215323-A

regulatory molecules which signals

measuring VEGF

Disorders; Orthopaedic

Implant)

US5266683-A

through receptor serine/threonine kinases

expression in fetal rat

Reconstruction;

to stimulate cellular responses. Its efects

calvaria cells. (Mol Cell

Parkinson's Disease;

include increased VEGF expression by

Endocrinol Jul. 20, 1999;

Periodontal Tissue

fetal calvaria cells and increased

153(1-2): 113-124).

Repair; Renal Failure;

dendritic outgrowth in cerebral cortical

Spinal Fusion; Stroke;

neurons.

Tooth Dentin

Regeneration; Long Bone

Nonunions

Osteoprotegrin

Locus

DE19654610-A1

Osteoprotegrin inhibits

Osteoprotegerin activity

Bone Disorders; Cancer

(Osteoclastogenesis

ID: 4982

WO9626217-A1

osteoclastogenesis and bone resorption,

may be assayed in vitro

Pain; Postmenopausal

Inhibiting Factor; Bone-

NP_002537

WO9626217-A1

and induces fibroblast proliferation.

using a coculture assay

Osteoporosis;

related protein)

for osteoclastogenesis, a

Rheumatoid Arthritis

bone resorption assay

using fetal long-bone

organ culture system, a

dentine resorption assay,

or a fibroblast

proliferation assay.

(FASEB J. 1998;

12: 845-854).

PAF acetyl-hydrolase

Locus

WO9509921

Platelet-activating factor acetylhydrolase

Plasma PAF

Asthma; Necrotizing

(Platelet activating factor

ID: 7941

US5656431

inactivates platelet-activating factor and

acetylhydrolase activity

Enterocolitis; Acute

(acetylhydrolase);

NP_005075

US5641669

related phospholipids.

may be determined by the

Enterocolitis; Adult

PAFASE)

XP_004492

US5605801

method of Stafforini et al.

Respiratory Distress

US5698403

(Stroke 1997

Syndrome; Allergic

US5847088

Dec; 28(12): 2417-20).

Asthma; inflammatory

Bowel Disorders;

Ischaemia/Reperfusion;

Pancreatitis; Sepsis-

Induced Systemic

Inflammatory Response

Syndrome; Solid Organ

Preservation; Type 1

Diabetes Mellitus;

Patched (hedgehog

Locus

WO9611260-A1

Patched, the receptor for Sonic

Patched function may be

Basal Cell Cancer; Brain

receptor)

ID: 5727

WO9953058-A1

hedgehog, is a tumour-suppressor which

assayed in vitro by

Cancer

NP_000255

EP879888-A2

controls developmental patterning and

measuring binding of its

XP_005574

growth.

ligand, Sonic hedgehog.

(Nature Nov. 14, 1996;

384(6605): 129-134).

PDGF (Becaplermin;

CAS-

EP282317-A

PDGF-B is a potent mitogen and

PDGF activity may be

Lower-Extremity Diabetic

PGDF-B; platelet-

165101-51-9

EP288307-A

transforming agent. It acts through the

assayed in vitro by

Neuropathic Ulcers;

derived growth factor;

LocusID: 5154

EP559234-A

PDGF receptor tyrosine kinase to exert

measuring its ability to

Decubitus Ulcer; Diabetic

RWJ 60235;

LocusID: 5155

EP622456-A

its mitogenic and transfoming effects.

induce tyrosine

Foot Ulcer; Venous

REGRANEX)

NP_002598

US5128321-A

phosphorylation on the

Stasis Ulcers; Periodontal

NP_002599

US5219739-A

PDGF receptor. (J Biol

Disease; Skin Ulcer;

XP_009997

US5474982-A

Chem May 25, 1989;

WO9116335-A

264(15): 8905-8912).

WO9320204-A

WO9405785-A

WO9405786-A

PEDF (Pigment

Locus

WO9533480-A

Pigment epithelium-derived factor is a

PEDF function may be

Amyotrophic Lateral

epithelium-derived

ID: 5176

WO9324529-A

noninhibitory member of the serpin

assayed in vitro using a

Sclerosis; Inflammatory

factor)

NP_002606

WO9904806-A1

family of serine protease inhibitors. It

neurite outgrowth assay.

Eye Diseases; Vascular

acts as a neurite-promoting factor by a

(J Biol Chem Oct. 27, 1995;

Eye Diseases;

mechanism other than serine protease

270(43): 25992-25999).

Degenerative Eye

inhibition.

Diseases; Dystrophic Eye

Disease; Neuropathies

Plasminogen activator

Locus

WO9013648-A

Plasminogen activator inhibitor I

Wallen, P., Biochemistry

Hemorrhage

inhibitor (Plasminogen

ID: 5054

WO9206203-A

regulates fibrinolysis by inhibiting the

of plasminogen. In:

activator inhibitor-1)

NP_000593

EP260757-A

conversion of inactive plasminogen to

Kline D. L., Reddy,

XP_004828

WO8801273-A

plasmin. Plasminogen activator

K. N. N., eds.

inhibitor I is a member of the serpin

Fibrinolysis. Boca

family of serine protease inhibitors.

Raton, FL: CRC Press,

1980: 1-25; Saksela, O.,

Rifkin, D. B., Cell-

associated plasminogen

activation: Regulation and

physiological functions. Annu Rev

Cell Biol 1988; 4: 93-126;

Womack C J, Ivey

F M, Gardner A W,

Macko R F, Fibrinolytic

response to acute exercise

in patients with

peripheral arterial disease.

Med Sci Sports Exerc

2001 Feb; 33(2): 214-9

Progenipoietin (PGP)

Genbank: Q62137

WO8604506-A

PGP is an engineered protein containing

PGP activity may be

Stem Cell Mobilization;

Genbank: P09919

WO8604605-A

both fetal liver tyrosine kinase-3 and

assayed in vitro by

Cancer;

PMID: 11164104

EP220520-A

granulocyte colony-stimulating factor

measuring agonism of

Myelosuppression;

EP217404-A

receptor agonist activities. It is a potent

fetal liver tyrosine kinase-

Neutropenia/

WO8701132-A

hematopoietic growth factor, capable of

3 and granulocyte colony-

Thrombocytopenia

generating multiple cell lineages,

stimulating factor. (Exp

Prevention

Hematol 2001 Jan;

29(1): 41-50).

Promegapoietin (PMP)

PMID: 10720698

PMP is an engineered protein chimera

PMP activity may be

Stem Cell Mobilization;

(IL-3/TPO receptor agonist) capable of

assayed in vitro by

Thrombocytopenia

inducing megakaryocytopoiesis in

measuing the ability to

CD43+ cells purified from bone marrow,

induce

mobilized peripheral blood progenitor

megakaryocytopoiesis in

cells, or umbilical cord.

CD43+ cells purified

from bone marrow,

mobilized peripheral

blood progenitor cells, or

umbilical cord. (J Hematother

1999 Apr; 8(2): 199-208).

Prosaptide (Prosaptide

Genbank: AAG31635

WO200105422.28

Prosaptide is a a neurotrophic peptide

Prosaptide efficacy can be

Diabetic Nephropathy;

TX 14; Prosaptide TX

WO9503821-A

(TXLIDNNATEEILY; where X equals

measured through

Pain; Central Nervous

152)

Pubmed: PMID:

WO9912559-A1

D-alanine) derived from prosaposin.

multiple dose regimens

System Disorders

9114068

using diabetic rats

(Anesthesiology 2000

Nov; 93(5): 1271-8).

Ranpirnase (Amphibian

Genbank: AF351207

US5955073-A

Ranpirnase is a pancreatic Rnase from

Ranpirnase activity in

Cancer; Autoimmune

ribonuclease A; P30;

WO9950398-A2

frog (Rana pipiens) eggs, which has low

vitro may be assayed by

Disorders; Brain Cancer;

ONCONASE)

enzyme activity, is not sensitive to

using ribonuclease and

HIV Infections Treatment;

ribonuclease inhibitor and is cytotoxic

cytotoxic activity assays.

Inflammatory Disorders;

to cancer cell lines and animals.

(J Mol Biol Apr. 19, 1996;

Mesothelioma; Non-

257(5): 992-1007).

Hodgkin's Lymphoma;

Pancreatic Cancer;

Prostate Cancer; Renal

Cancer;

Relaxin (Human relaxin;

LocusID: 6013

EP101309

Relaxin is a peptide hormone

Relaxin activity may be

Scleroderma; Infertility;

SUN Y8002; CONXN)

LocusID: 6019

EP112149

synthesized in the corpora lutea of

assayed in vitro using an

Congestive Heart Failure;

NP_008842

EP303033

ovaries during pregnancy and is released

inhibition of KCl-induced

Labour Disorders;

NP_005050

US7783046

into the blood stream prior to

rat uterine contractions in

Peripheral Arterial

XP_011804

WO9013659

parturition. Its major biological effect is

vitro assay. (Can J

Disorders; Pulmonary

XP_005512

to remodel the mammalian reproductive

Physiol Pharmacol 1981

Fibrosis; Pulmonary

tract to facilitate the birth process.

May; 59(5): 507-12).

Hypertension

Retinal S-antigen

Genbank: AAA30378

WO9101333-A

Retinal S-Antigen is a photoreceptor

Retinal S-Antigen

Uveitis

(Retinal Arrestin;

protein which binds to phosphorylated

activity may be assayed

AI300)

rhodopsins and plays a role in

in vitro by measuring

deactivating the phototransduction

activity of cyclic GMP-

cascade.

gated channels in rod

photoreceptor cells. (J

Biol Chem Nov. 25, 1994;

269(47): 29765-29770).

RhLH (Human

LocusID: 1081

WO8810270

RhLH is a pituitary hormone dimer

RhLH activity may be

Infertility

luteinizing hormone;

LocusID: 3972

EP735139

consisting alpha and beta subunits that

assayed in vitro by

Lutropin; r-hLH; Lhadi;

NP_000726

US6083706

are associated noncovalently. Its main

measuring Fura-2

LUVERIS)

NP_000885

WO8607383-A

effects are in the maturation of oocytes

fluorescence in isolated

XP_011444

WO9116922-A

and the secretion of estrogen and

porcine thecal cells in

XP_009418

WO9116922-A

progesterone by ovarian follicles.

response to RhLH

WO9858957-A2

stimulation.

WO9858957-A2

(Endocrinology 2000

Jun; 141(6): 2220-2228).

Saruplase (CG 4509;

CAS-99149-

EP154272-A

Saruplase is an unglucosylated single-

Saruplase activity may be

Myocardial Infarction

scu-PA;

95-8

EP139447-A

chain recombinant urokinase-type

measured in vitro using a

RESCUPLASE)

Genbank: CAA01515

EP231883-A

plasminogen activator.

plasminogen cleavage

EP288435-A

assay. (J Biol Chem

WO8901513-A

Jan. 18, 2001; [epub ahead

EP620279-A

of print]).

WO200026353-A1

Serine protease

Genbank: AAA46629.1

WO9630042-A2

Serine protease inhibitor-1 (myxoma

Serine protease inhibitor-

Atherosclerosis; Renal

inhibitor-1 (Serine

WO9527503-A1

virus derived) is an antiinflammatory

1 (myxoma virus derived)

Transplant Rejection;

protease inhibitor-1

serine protease inhibitor.

activity can be measured

Reperfusion Injury;

(myxoma virus))

through an antigen

Rheumatoid Arthritis

induced arthritis (AIA)

model of chronic

inflammation (J

Rheumatol 1996

May; 23(5): 878-82).

Soluble complement

LocusID: 1378

EP351246-A

Complement receptor type 1 (C3b/C4b

Complement receptor

Myocardial Infarction;

receptor type 1 (TP10;

NP_000564

EP512733-A

receptor) is the receptor for C3b-and

type 1 may be assayed in

Acute Respiratory

sCR1; BRL 55736)

NP_000642

US5041376-A

C4b-coated ligands. It lacks

vitro by measuring C3b

Distress Syndrome; Adult

US5200340-A

transmembrane and cytoplasmic

and C4b binding. (J Exp

Respiratory Distress

US5714372-A

domains and inhibits C3 and C5

Med Nov. 1, 1988;

Syndrome; Allotransplant

US5981481-A

convertase activity by preferentially

168(5): 1699-1717).

Rejection; Disseminated

WO8900197-A

binding C4b and C3b.

Intravascular Coagulation;

WO8909220-A

Lung Transplant

WO9105047-A

Rejection; Multiple

WO9400571-A

Sclerosis; Reperfusion

Injury; Rheumatoid

Arthritis; Systemic Lupus

Erythematosus;

Xenotransplant Rejection

Sonic hedgehog

LocusID: 6469

WO9518856

Sonic hedgehog plays a central role in

Sonic hedgehog function

Parkinson's Disease;

(hedgehog)

NP_000184

WO9920298

patterning of the embryo. It occurs as an

may be assayed in vitro

Neurological Disorders;

XP_004942

inactive precursor which must be

by measuring its binding

Alopecia

proteolytically cleaved and modified to

to patched. (Nature 1996

become active as a signalling molecule.

Nov. 14; 384(6605): 129-134).

The receptor for Sonic hedgehog is Patched,

a multi-pass transmembrane

protein, and downstream targets of

Sonic hedgehog signalling are transcription

factors like Gli3.

Staphylokinase

Genbank: CAA29822

WO9211356

Staphylokinase is a single domain

Staphylokinase activity

Myocardial Infarction;

(SakSTAR; Sak42D)

Genbank: CAA01335

WO9313209

protein that proteolytically activates

may be measured in vitro

Arterial Occlusive

plasminogen.

using a plasminogen

Disorders; Stroke

cleavage assay. (J Biol

Chem Jan. 18, 2001; [epub

ahead of print]).

Stem cell factor (Kit

CAS-

EP423980-A

SCF is substantially necessary for mast

SCF activity may be

Mobilization Of

ligand; Ancestim; AMJ

163545-26-4

EP676470-A1

cell survival and induces marginal mast

assayed by measuring

Progenitor Cells Before

9302; human SCF;

NP_003985

EP423980-A

cell proliferation in vitro. In the presence

mast cell survival,

PBPCT In Breast Cancer;

STEMGEN)

WO9200376-A

of SCF, mast cells predominantly

proliferation, or

In Conjunction With

produce pro-inflammatory cytokines

production of pro-

Neupogen; Stem Cell

including tumor necrosis factor (TNF)-

inflammatory cytokines.

Mobilization; Anemia;

alpha, IL-1beta, IL-6, IL-8, IL-16, and

(Immunol Rev 2001 Feb;

Xenotransplant Rejection

IL-18.

179: 57-60).

Streptokinase

CAS-9002-

US6087332

Streptokinase is a 3 domain

Streptokinase activity

Myocardial Infarction;

(STREPTASE;

01-1

(alpha, beta, gamma) molecule, which

may be measured in vitro

Blood Clots; Pulmonary

KABIKINASE)

Genbank: E03308

non-proteolytically activates human (h)-

using a plasminogen

Embolism

plasminogen and protects plasmin from

activation assay. (J Biol

inactivation by alpha2-antiplasmin.

Chem Jan. 18, 2001; [epub

ahead of print]).

Superoxide dismutase

CAS-9016-

EP492447-A

Copper zinc superoxide dismutase is an

Superoxide dismutase

Asthma;

(Pegorgotein; Peg-SOD;

01-7

EP676472-A1

intracellular protein which catalyzes

activity may be assayed

Bronchopulmonary

PC-SOD; Orgotein;

LocusID: 6647

US5540911-A

dismutation of superoxide to oxygen

in vitro using a

Dysplasia In Prematurity;

OXSODROL;

LocusID: 6648

US5683864-A

and hydrogen peroxide.

superoxide dismutase

Burns; Eye Disorders;

DISMUTEC;

LocusID: 6649

US5688688-A

assay. (Nucleic Acids Res

Head Injuries; Infant

OXSODROL;

NP_000445

US5712087-A

Mar. 25, 1985;

Respiratory Distress

ONTOSEIN;

NP_000627

US5780024-A

13(6): 2017-34).

Syndrome; Inflammatory

ARTROLASI;

NP_003093

US5849290-A

Disorders; Kidney

INTERCEPTOR;

XP_009723

US6107070-A

Disorders; Myocardial

ORGO; OXINORM;

XP_004242

WO8701387-A

Infarction; Ischemic Heart

PEROXINORM;

XP_003578

WO9009434-A

Disorders; Reperfusion

SEROSOD)

WO9010694-A

Injury; Respiratory

WO9104315-A

Disorders; Stroke;

WO9414950-A

Autoimmune Disorders;

WO9419493-A

Acute Lung Injury; HIV

Infections Treatment;

Amyotrophic Lateral

Sclerosis; Respiratory

Syncytial Virus; Cystits;

Radioprotection;

Rheumatic Disorders

T1/ST2 receptor

Genbank: P14719

US5180812-A

T1/ST2 is a member of the IL-1R

T1/ST2 function may be

Asthma; Allergies

(T1/ST2 receptor)

US5488032-A

family, preferentially expressed on the

assayed in vitro using a

EP381296-A

surface of Th2 cells, and it plays an

Th2 cell activation assay.

EP1033401-A2

important role in the activation of Th2

(J Immunol Mar. 1, 2001;

EP381296-A

cells.

166(5): 3143-3150).

TGF Beta 1

LocusID:

EP293785-A

Regulates cell proliferation,

TGF beta 1 activity may

Alzheimer's Disease;

(Transforming growth

7040

WO8912101-A

differentiation, and apoptosis in

be assayed in vitro by

Atherosclerosis; Cancer;

factor-beta)

NP_000651

US4886747-A

numerous cell types.

measuring induction of

Skin Disorders; Systemic

XP_008912

US5409896-A

renal fibroblast

Lupus Erythematosus;

US5482851-A

proliferation via induction

Transplant Rejection

US5284763-A

of FGF-2. (Kidney Int

US5801231-A

2001 Feb; 59(2): 579-592).

TGF Beta 2

LocusID: 7042

WO8912101

Transforming growth factor-beta 2

TGF Beta 2 function may

Chronic Wounds;

(Transforming Growth

NP_003229

WO9014360

(TGF-beta 2) has been found to inhibit

be assayed in vitro by

Mucositis; Age-Related

Factor Beta 2; cetermin;

XP_001754

US5409896

inducible nitric oxide synthase (iNOS)

measuring inhibition of

Macular Degeneration;

BetaKine)

US5801231

gene transcription, esp. in interleukin-1-

iNOS transcription.

Autoimmune Disorders;

US5284763

beta (IL1-beta) stimulated rat smooth

(Inflamm Res 1997 Sep;

Cancer; Diabetic Foot

muscle cells, and at a dose which does

46(9): 327-331).

Ulcer; Eye Disorders;

not inhibit consitutive NOS.

Multiple Sclerosis;

Postmenopausal

Osteoporosis;

Rheumatoid Arthritis;

Skin Disorders;

Transplant Rejection;

TGF Beta 3

LocusID: 7043

WO9840747-A1

Transforming growth factor-beta 3 is a

TGF-beta 3 can be

Chronic Wounds; Oral

(Transforming Growth

NP_003230

WO9200318-A

cytokine which transmits mitogenic

assayed for stimulation of

Mucositis;

Factor Beta 3; CGP-

XP_007417

US5409896-A

signals through transmembrane

production of

Radioprotection

46614)

US5801231-A

serine/threonine kinases. TGF beta 3 is

prostaglandin E2 (PGE2)

US5409896-A

required for normal development of the

and bone resorption in

US5284763-A

lung and palate.

neonatal mouse calvaria

WO9119513-A

in organ culture (PNAS

USA 1985 Jul;

82(13): 4535-4538), or

stimulation of the

synthesis of collagen,

osteopontin, osteonectin,

and alkaline phosphatase,

and the ability to

stimulate replication in

osteoblast-like cells (J

Biol Chem 1987;

262: 2869, J Biol Chem

1988; 263: 13916, J Cell

Biol 1988; 106: 915, J

Cell Physiol 1987; 133: 426,

Endocrinology 1987; 121: 212,

Endocrinology 1986;

119: 2306, and J Cell

Biol 1987; 105: 457).

Thrombopoietin

LocusID: 7066

WO9521626-A1

Thrombopoietin binds to the c-Mpl

Thrombopoietin (TPO)

Thromocytopenia;

(Pegacaristim; KRN

NP_000451

EP675201-A

receptor and regulates megakaryocyte

can be assayed to

Chemoprotection

9000; Megakaryocyte

XP_002815

GB2285446-A

development.

determine regulation of

growth and development

WO9628181-A1

growth and differentiation

factor; PEG

US6099830-A

of megakaryocytes. (Mol

thrombopoietin; rhTPO)

Cell Biol 2001 Apr;

21(8): 2659-2670; Exp

Hematol 2001 Jan;

29(1): 51-58; Leukemia

2000 Oct; 14(10): 1751-1756).

Tie-2 (Tek; tunica

LocusID: 7010

WO9314124

Tie-2/Tek, the angiopoietin receptor

Tie-2/Tek function may

Cancer

interna endothelial-cell

NP_000450

WO9400469

tyrosine kinase, mediates intracellular

be assayed by measuring

kinase)

XP_005480

WO9513387

signalling and angiogenic responses

its phosphorylation in

following stimulation by angiopoietin.

reponse to stimulation by

angiopoietin. (Int

Immunol 1998 Aug;

10(8): 1217-1227).

Tissue Factor Pathway

LocusID: 7035

WO9521601

Tissue factor pathway inhibitor is a

Tissue factor pathway

Sepsis; Coagulation

Inhibitor (Tifacogin;

NP_006278

WO9502059

lipoprotein-associated coagulation

inhibitor may be assayed

Disorders; Reperfusion

LACI; SC 59735)

XP_002672

WO9518830

inhibitor. It is a Kunitz-type protease

for inactivation of factor

Injury; Atherosclerosis;

US5212091

inhibitor that inhibits fibrin clot

Xa and inhibition of the

EP318451

formation.

VIIa-tissue factor complex

of the extrinsic

coagulation pathway. (J

Biol Chem May 5, 1998;

263(13): 6001-6004;

Thromb Haemost 1998;

79(2): 306-309).

TNFAlpha (MHR 24;

LocusID: 7124

WO8604606-A

TNF plays a central role in the

TNF binding protein

Cancer; CNS Cancer;

PT 050; Sertenef;

NP_000585

WO8806625-A

pathophysiology of sepsis. High levels

activity may be assayed

Lymphoma; Skin Cancer;

Sonermin; TNF-SAM2;

XP_011402

WO9102540-A

of TNF alpha correlates with increased

by measuring inhibition

Urogenital Cancer;

PEG-TNF; TIENEF)

US5853977-A

disease severity in severe bacterial

of PIP5K activation in

WO8603751-A

infection and malaria TNF alpha

response to TNF

US5487984-A

signaling may lead to activation of NF

stimulation of HeLa cells.

US5853977-A

kappa B and induction of apoptosis.

(J Biol Chem Feb. 28, 1997;

272(9): 5861-5870).

TNF binding protein

NP_001056

WO9319777-A

TNF binding protein inhibits activation

TNF binding protein

Rheumatoid Arthritis;

(TBP I; TNFbp; TNF-

EP939121-A2

of the TNF-receptor by competing for

activity may be assayed

Cardiac Reperfusion

BP-1; soluble TNF-

US6143866-A

TNF binding.

by measuring inhibition

Injury; Autoimmune

alpha receptor domain)

WO9207076-A

of PIP5K activation in

Disorders; Crohn's

response to TNF

Disease; Malaria;

stimulation of HeLa cells.

Reperfusion Injury; Septic

(J Biol Chem Feb. 28, 1997;

Shock;

272(9): 5861-5870).

TNF Receptor

CAS-

EP417563-A

Tumor Necrosis Factor receptor

TNF receptor activity

Rheumatoid Arthritis;

(Etanercept; p75TNFR-

185243-69-0

EP418014-A

mediates proinflammatory cellular

may be assayed by

Cachexia; Heart Failure;

Ig; p55TNFR-Ig; rhu

LocusID: 7132

EP648783-A

responses in response to TNF

measuring increases of

HIV 1 Infections; Juvenile

TNFR-Fc; Lenercept;

LocusID: 7133

EP939121-A2

stimulation.

PIP5K activation in

Rheumatoid Arthritis;

sTNF R1; ENBREL;

NP_001056

US6143866-A

response to TNF

Psoriasis; Psoriatic

TENEFUSE)

NP_001057

WO9207076-A

stimulation of HeLa cells.

Arthritis; Septic Shock;

XP_006950

WO9319777-A

(J Biol Chem Feb. 28, 1997;

Transplant Rejection;

XP_001743

WO9406476-A

272(9): 5861-5870).

Allergic Asthma

WO9849305-A1

t-PA (Alteplase;

CAS-

EP178105-A

Tissue-type plasminogen activator;

Wallen, P., Biochemistry

Embolism; Myocardial

Lanoteplase;

105857-23-6

EP351246-A

serine protease that converts inactive

of plasminogen. In:

Infarction; Stroke;

Monteplase;

CAS-

GB2173804-A

plasminogen to plasmin

Kline D. L., Reddy,

Thrombosis; Congestive

Pamiteplase; Reteplase;

171870-23-8

US4963357-A

K. N. N., eds.

Heart Failure; Ischemic

Tenecteplase; E 6010;

CAS-

US5037752-A

Fibrinolysis. Boca

Heart Disorders; Coronary

SUN 9216; BMS

156616-23-8

US5041376-A

Raton, FL: CRC Press,

Restenosis

200980; FEX1; nPA;

CAS-

US5200340-A

1980: 1-25; Saksela, O.,

Oneplas; Desmoteplase;

151912-42-4

US5504001-A

Rifkin, D. B., Cell-

NO-tPA; ACTIVASE;

CAS-

US5714372-A

associated plasminogen

ACTIVACIN; GRTPA;

133652-38-7

US5985607-A

activation: Regulation

CLEACTOR;

CAS-

WO8900197-A

and physiological

SOLINASE;

191588-94-0

functions. Annu Rev

RETAVASE; TNKase;

LocusID: 5327

Cell Biol 1988; 4: 93-126;

RAPILYSIN;

NP_000921

Womack C J, Ivey

METALYSE)

NP_000922

FM, Gardner A W,

XP_005024

Macko R F, Fibrinolytic

response to acute exercise

in patients with

peripheral arterial disease.

Med Sci Sports Exerc

2001 Feb; 33(2): 214-9

Trophoblast interferon

Genbank: A53746

WO9410313-A

Involved in placental cell growth and

IFN-tau activity may be

HIV Infections Treatment;

(interferon tau; IFN-tau;

WO9635789-A1

differentiation, as well protecting the

assayed in vitro by

Multiple Sclerosis

Trophoblastin)

WO9739127-A1

fetus in viral environments

measuring IFN-tau

induced production of an

acidic 70 kD protein in

cultured explants of

endometrium prepared

from ewes on day 13 of

the estrous cycle. (Mol

Endocrinol 1990 Oct;

4(10): 1506-1514).

Troponin 1

LocusID: 7135

WO9730085

Troponin I is a regulatory protein which

Troponin I activity may

Cancer Metasteses;

NP_003272

US5834210

prevents actin and myosin interaction in

be assayed in vitro using

Diabetic Retinopathy;

XP_001918

US6072040

resting muscle tissue. Troponin I an

a myofibril binding

Eye Disorders; Macular

LocusID: 7136

US6060278

inhibitory subunit of troponin.

assay. (J Muscle Res Cell

Degeneration; Solid

Motil 1999 Nov;

Tumors

20(8): 755-760).

Urate oxidase (Uricase;

LocusID: 7377

US5541098

Urate oxidase is an enzyme which

Urate oxidase activity

Chemoprotection;

Peg Uricase;

Genbank: S94095

catalyzes the oxidation of uric acid.

may be assayed in vitro

Prevention And

Rasburicase;

using an assay for uricase-

Treatment Of

FASTURTEC;

catalyzed oxidation of

Chemotherapy-Related

PERICASE)

uric acid (Anal Chem

Uricemia; Gout

May 15, 1999;

71(10): 1928-1934); or by

immunoassay of

recombinant urate oxidase

(J Pharm Sci 1996 Sep;

85(9): 955-959).

Urokinase (Pro-

CAS-9039-

EP154272-A

Urokinase acts as a plasminogen

Urokinase activity may

Catheter Clearance;

urokinase; FCE 26177;

53-6

EP231883-A

activator involved in blood clotting

be measured in vitro

Coronary Restenosis;

FCE 27485; GE 0493;

LocusID: 5328

WO8901513-A

regulation. It is a serine protease that

using a plasminogen

Diabetic Retinopathy;

Lys300His mutant pro-

NP_002649

WO8604351-A

cleaves plasminogen to form plasmin.

cleavage assay. Sazonova

Myocardial Infarction;

urokinase;

XP_011861

EP139447-A

et al. (J Biol Chem Jan. 18, 2001

Thrombosis; Vitreous

ABBOKINASE;

US5648253-A

[electronic

Haemorrhage; Peripheral

NASARUPLASE;

publication prior to

Vascular Disorders;

THROMBOLYSE;

print]).

Stroke

PROLYSE)

VEGF-1 (VEGF-121)

LocusID: 7422

US5726152

VEGF-1 is a growth factor which

VEGF-1 activity may be

Cardiovascular Disease;

LocusID: 7423

US5840693

induces endothelial cell proliferation and

assayed in vitro using an

Vascular Disorders;

LocusID: 7424

US5932540

vascular permeability.

endothelial cell

Fracture Treatment;

NP_003367

US5994300

proliferation assay. (Proc

NP_003368

US6037329

Natl Acad Sci U.S.A. 1989

NP_005420

WO9013649

Feb; 86(3): 802-806).

XP_004512

WO9626736

XP_006539

WO9627007

XP_003456

WO9639421

WO9639515

WO9705250

WO9709427

Viscumin (mistletoe

Genbank: CAA03513

EP751221

A recombinant, galactoside specific

Activity of Viscumin

Cancer

lectin)

EP884388

mistletoe lectin (rML), which activates

may be assayed using

granulocytes and neutrophils

granulocyte and

neutrophil activation

assays. (Anticancer Res

1999 Jul-Aug;

19(4B): 2925-2928; and J

Leukoc Biol 2000 Dec;

68(6): 845-853).

In preferred embodiments, the albumin fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity corresponding to the therapeutic activity and/or biologic activity of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein listed in the corresponding row of Table 1. (See, e.g., the “Biological Activity” and “Therapeutic Protein X” columns of Table 1.) In further preferred embodiments, the therapeutically active protein portions of the albumin fusion proteins of the invention are fragments or variants of the reference sequence cited in the “Exemplary Identifier” column of Table 1, and are capable of the therapeutic activity and/or biologic activity of the corresponding Therapeutic protein disclosed in “Biological Activity” column of Table 1.

Polypeptide and Polynucleotide Fragments and Variants

Fragments

The present invention is further directed to fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.

Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the Therapeutic protein, albumin protein, and/or albumin fusion protein, other Therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of polypeptides with N-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (e.g., a Therapeutic protein as disclosed in Table 1). In particular, N-terminal deletions may be described by the general formula m−q, where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, fragments of serum albumin polypeptides corresponding to an albumin protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (i.e., serum albumin). In particular, N-terminal deletions may be described by the general formula m-585, where 585 is a whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO: 18), and m is defined as any integer ranging from 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Moreover, fragments of albumin fusion proteins of the invention, include the full length albumin fusion protein as well as polypeptides having one or more residues deleted from the amino terminus of the albumin fusion protein. In particular, N-terminal deletions may be described by the general formula m−q, where q is a whole integer representing the total number of amino acid residues in the albumin fusion protein, and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the N-terminus or C-terminus of a reference polypeptide (e.g., a Therapeutic protein and/or serum albumin protein) results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) and/or Therapeutic activities may still be retained. For example the ability of polypeptides with C-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the N-terminal and/or C-terminal residues of a reference polypeptide retains Therapeutic activity can readily be determined by routine methods described herein and/or otherwise known in the art.

The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., a Therapeutic protein referred to in Table 1). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1). Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., serum albumin). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to 584, where 584 is the whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO:18) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Moreover, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of an albumin fusion protein of the invention. In particular, C-terminal deletions may be described by the general formula 1−n, where n is any whole integer ranging from 6 to q−1, and where q is a whole integer representing the total number of amino acid residues in an albumin fusion protein of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m−n of a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1, or serum albumin (e.g., SEQ ID NO: 18), or an albumin fusion protein of the invention) where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference polypeptide sequence (e.g., a Therapeutic protein, serum albumin protein or an albumin fusion protein of the invention) set forth herein, or fragments thereof. In preferred embodiments, the application is directed to proteins comprising polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to reference polypeptides having the amino acid sequence of N- and C-terminal deletions as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a Therapeutic activity and/or functional activity (e.g. biological activity) of the polypeptide sequence of the Therapeutic protein or serum albumin protein of which the amino acid sequence is a fragment.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Variants

“Variant” refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.

As used herein, “variant”, refers to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin portion of an albumin fusion protein of the invention, or albumin fusion protein differing in sequence from a Therapeutic protein (e.g. see “therapeutic” column of Table 1), albumin protein, and/or albumin fusion protein of the invention, respectively, but retaining at least one functional and/or therapeutic property thereof (e.g., a therapeutic activity and/or biological activity as disclosed in the “Biological Activity” column of Table 1) as described elsewhere herein or otherwise known in the art. Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention, and/or albumin fusion protein of the invention. Nucleic acids encoding these variants are also encompassed by the invention.

The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., an amino acid sequence disclosed in the “Exemplary Identifier” column of Table 1, or fragments or variants thereof), albumin proteins (e.g., SEQ ID NO:18 or fragments or variants thereof) corresponding to an albumin protein portion of an albumin fusion protein of the invention, and/or albumin fusion proteins of the invention. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding an amino acid sequence of the invention under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6×Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2×SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g., hybridization to filter bound DNA in 6×sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides are also encompassed by the invention.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of an albumin fusion protein of the invention or a fragment thereof (such as the Therapeutic protein portion of the albumin fusion protein or the albumin portion of the albumin fusion protein), can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

The variant will usually have at least 75% (preferably at least about 80%, 90%, 95% or 99%) sequence identity with a length of normal HA or Therapeutic protein which is the same length as the variant. Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated by reference) which are tailored for sequence similarity searching.

The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al., (Nature Genetics 6: 119-129 (1994)) which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and −4, respectively. Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host, such as, yeast or E. coli).

In a preferred embodiment, a polynucleotide encoding an albumin portion of an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells. In further preferred embodiment, a polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells. In a still further preferred embodiment, a polynucleotide encoding an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.

In an alternative embodiment, a codon optimized polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the Therapeutic protein under stringent hybridization conditions as described herein. In a further embodiment, a codon optimized polynucleotide encoding an albumin portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein. In another embodiment, a codon optimized polynucleotide encoding an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the Therapeutic protein portin or the albumin protein portion under stringent hybridization conditions as described herein.

In an additional embodiment, polynucleotides encoding a Therapeutic protein portion of an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of that Therapeutic protein. In a further embodiment, polynucleotides encoding an albumin protein portion of an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein. In an alternative embodiment, polynucleotides encoding an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of a Therapeutic protein portion or the albumin protein portion.

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which have a functional activity (e.g., biological activity and/or therapeutic activity). In highly preferred embodiments the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity, such as that disclosed in the “Biological Activity” column in Table 1) that corresponds to one or more biological and/or therapeutic activities of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.

In preferred embodiments, the variants of the invention have conservative substitutions. By “conservative substitutions” is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) polypeptides containing substitutions of one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino acid residues having a substituent group, or (iii) polypeptides which have been fused with or chemically conjugated to another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) polypeptide containing additional amino acids, such as, for example, an IgG Fc fusion region peptide. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of a Therapeutic protein described herein and/or human serum albumin, and/or albumin fusion protein of the invention, wherein the fragments or variants have 1-5,5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the invention.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Functional Activity

“A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with the full-length, pro-protein, and/or mature form of a Therapeutic protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.

“A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a Therapeutic protein of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).

In preferred embodiments, an albumin fusion protein of the invention has at least one biological and/or therapeutic activity associated with the Therapeutic protein (or fragment or variant thereof) when it is not fused to albumin.

The albumin fusion proteins of the invention can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, albumin fusion proteins may be assayed for functional activity (e.g., biological activity or therapeutic activity) using the assay referenced in the “Exemplary Activity Assay” column of Table 1. Additionally, one of skill in the art may routinely assay fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, for activity using assays referenced in its corresponding row of Table 1. Further, one of skill in the art may routinely assay fragments of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention, for activity using assays known in the art and/or as described in the Examples section below.

For example, in one embodiment where one is assaying for the ability of an albumin fusion protein of the invention to bind or compete with a Therapeutic protein for binding to an anti-Therapeutic polypeptide antibody and/or anti-albumin antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In a preferred embodiment, where a binding partner (e.g., a receptor or a ligand) of a Therapeutic protein is identified, binding to that binding partner by an albumin fusion protein containing that Therapeutic protein as the Therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of an albumin fusion protein of the present invention to bind to a substrate(s) of the Therapeutic polypeptide corresponding to the Therapeutic portion of the albumin fusion protein of the invention can be routinely assayed using techniques known in the art.

In an alternative embodiment, where the ability of an albumin fusion protein of the invention to multimerize is being evaluated, association with other components of the multimer can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., supra.

In addition, assays described herein (see Examples and Table 1) and otherwise known in the art may routinely be applied to measure the ability of albumin fusion proteins of the present invention and fragments, variants and derivatives thereof to elicit biological activity and/or Therapeutic activity (either in vitro or in vivo) related to either the Therapeutic protein portion and/or albumin portion of the albumin fusion protein of the present invention. Other methods will be known to the skilled artisan and are within the scope of the invention.

Albumin

As described above, an albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion or chemical conjugation.

The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, “albumin and “serum albumin” are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

As used herein, “albumin” refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, “albumin” refers to human albumin or fragments thereof (see EP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form of human albumin as shown in FIG. 15 and SEQ ID NO: 18, or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

In preferred embodiments, the human serum albumin protein used in the albumin fusion proteins of the invention contains one or both of the following sets of point mutations with reference to SEQ ID NO:18: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gln, and Lys-414 to Gln (see, e.g., International Publication No. WO95/23857, hereby incorporated in its entirety by reference herein). In even more preferred embodiments, albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations have improved stability/resistance to yeast Yap3p proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.

As used herein, a portion of albumin sufficient to prolong the therapeutic activity or shelf-life of the Therapeutic protein refers to a portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the Therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the shelf-life in the non-fusion state. The albumin portion of the albumin fusion proteins may comprise the full length of the HA sequence as described above or as shown in FIG. 15, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA. For instance, one or more fragments of HA spanning the first two immunoglobulin-like domains may be used.

The albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA. The Therapeutic protein portion of the albumin fusion proteins of the invention may also be variants of the Therapeutic proteins as described herein. The term “variants” includes insertions, deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active site, or active domain which confers the therapeutic activities of the Therapeutic proteins.

In particular, the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419). The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the Therapeutic protein portion.

Preferably, the albumin portion of an albumin fusion protein of the invention comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or all of the adjacent linker is preferably used to link to the Therapeutic protein moiety.

Albumin Fusion Proteins

The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. As used herein, “albumin fusion protein” refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or chemical conjugation to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may be referred to as a “portion”, “region” or “moiety” of the albumin fusion protein.

In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.

In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein.

In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin.

Preferably, the albumin fusion protein comprises HA as the N-terminal portion, and a Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion, and a Therapeutic protein as the N-terminal portion may also be used.

In other embodiments, the albumin fusion protein has a Therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are the same Therapeutic proteins. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins. In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins which may be used to treat or prevent the same disease, disorder, or condition (e.g. as listed in the “Preferred Indication Y” column of Table 1). In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins which may be used to treat or prevent diseases or disorders (e.g. as listed in the “Preferred Indication Y” column of Table 1) which are known in the art to commonly occur in patients simultaneously.

In addition to albumin fusion protein in which the albumin portion is fused N-terminal and/or C-terminal of the Therapeutic protein portion, albumin fusion proteins of the invention may also be produced by inserting the Therapeutic protein or peptide of interest (e.g., Therapeutic protein X as diclosed in Table 1) into an internal region of HA. For instance, within the protein sequence of the HA molecule a number of loops or turns exist between the end and beginning of α-helices, which are stabilized by disulphide bonds (see FIGS. 9-11). The loops, as determined from the crystal structure of HA (FIG. 13) (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E71 and 1UOR) for the most part extend away from the body of the molecule. These loops are useful for the insertion, or internal fusion, of therapeutically active peptides, particularly those requiring a secondary structure to be functional, or Therapeutic proteins, to essentially generate an albumin molecule with specific biological activity.

Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the HA molecule and in which all possible combinations of amino acids are represented.

Such library(s) could be generated on HA or domain fragments of HA by one of the following methods:

(a) randomized mutation of amino acids within one or more peptide loops of HA or HA domain fragments. Either one, more or all the residues within a loop could be mutated in this manner (for example see FIG. 10a);

(b) replacement of, or insertion into one or more loops of HA or HA domain fragments (i.e., internal fusion) of a randomized peptide(s) of length Xn (where X is an amino acid and n is the number of residues (for example see FIG. 10b);

The HA or HA domain fragment may also be made multifunctional by grafting the peptides derived from different screens of different loops against different targets into the same HA or HA domain fragment.

In preferred embodiments, peptides inserted into a loop of human serum albumin are peptide fragments or peptide variants of the Therapeutic proteins disclosed in Table 1. More particulary, the invention encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids in length inserted into a loop of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the N-terminus of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the C-terminus of human serum albumin.

Generally, the albumin fusion proteins of the invention may have one HA-derived region and one Therapeutic protein-derived region. Multiple regions of each protein, however, may be used to make an albumin fusion protein of the invention. Similarly, more than one Therapeutic protein may be used to make an albumin fusion protein of the invention. For instance, a Therapeutic protein may be fused to both the N- and C-terminal ends of the HA. In such a configuration, the Therapeutic protein portions may be the same or different Therapeutic protein molecules. The structure of bifunctional albumin fusion proteins may be represented as: X-HA-Y or Y-HA-X.

For example, an anti-BLyS™ scFv-HA-IFNα-2b fusion may be prepared to modulate the immune response to IFNα-2b by anti-BLyS™ scFv. An alternative is making a bi (or even multi) functional dose of HA-fusions e.g. HA-IFNα-2b fusion mixed with HA-anti-BLy™ scFv fusion or other HA-fusions in various ratio's depending on function, half-life etc.

Bi- or multi-functional albumin fusion proteins may also be prepared to target the Therapeutic protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of HA.

As an alternative to the fusion of known therapeutic molecules, the peptides could be obtained by screening libraries constructed as fusions to the N-, C- or N- and C-termini of HA, or domain fragment of HA, of typically 6, 8, 12, 20 or 25 or Xn (where X is an amino acid (aa) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented. A particular advantage of this approach is that the peptides may be selected in situ on the HA molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to HA.

Additionally, the albumin fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the Therapeutic protein portion, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids such that it is flexible or more rigid.

The linker sequence may be cleavable by a protease or chemically to yield the growth hormone related moiety. Preferably, the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases.

Therefore, as described above, the albumin fusion proteins of the invention may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein, peptide or polypeptide sequence, and not necessarily the same Therapeutic protein, L is a linker and R2 is a serum albumin sequence.

In preferred embodiments, Albumin fusion proteins of the invention comprising a Therapeutic protein have extended shelf life compared to the shelf life the same Therapeutic protein when not fused to albumin. Shelf-life typically refers to the time period over which the therapeutic activity of a Therapeutic protein in solution or in some other storage formulation, is stable without undue loss of therapeutic activity. Many of the Therapeutic proteins are highly labile in their unfused state. As described below, the typical shelf-life of these Therapeutic proteins is markedly prolonged upon incorporation into the albumin fusion protein of the invention.

Albumin fusion proteins of the invention with “prolonged” or “extended” shelf-life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the unfused full-length Therapeutic protein. When the Therapeutic protein portion of the albumin fusion protein is an analog, a variant, or is otherwise altered or does not include the complete sequence for that protein, the prolongation of therapeutic activity may alternatively be compared to the unfused equivalent of that analog, variant, altered peptide or incomplete sequence. As an example, an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point.

Shelf-life may also be assessed in terms of therapeutic activity remaining after storage, normalized to therapeutic activity when storage began. Albumin fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90% or more of the therapeutic activity of the equivalent unfused Therapeutic protein when subjected to the same conditions. For example, as discussed in Example 1, an albumin fusion protein of the invention comprising hGH fused to the full length HA sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions.

Expression of Fusion Proteins

The albumin fusion proteins of the invention may be produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line. Preferably, the polypeptide is secreted from the host cells. We have found that, by fusing the hGH coding sequence to the HA coding sequence, either to the 5′ end or 3′ end, it is possible to secrete the albumin fusion protein from yeast without the requirement for a yeast-derived pro sequence. This was surprising, as other workers have found that a yeast derived pro sequence was needed for efficient secretion of hGH in yeast.

For example, Hiramatsu et al. (Appl Environ Microbiol 56:2125 (1990); Appl Environ Microbiol 57:2052 (1991)) found that the N-terminal portion of the pro sequence in the Mucor pusillus rennin pre-pro leader was important. Other authors, using the MFα-1 signal, have always included the MFα-1 pro sequence when secreting hGH. The pro sequences were believed to assist in the folding of the hGH by acting as an intramolecular chaperone. The present invention shows that HA or fragments of HA can perform a similar function.

Hence, a particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast-derived pro sequence between the signal and the mature polypeptide.

The Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.

Conjugates of the kind prepared by Poznansky et al., (FEBS Lett. 239:18 (1988)), in which separately-prepared polypeptides are joined by chemical cross-linking, are not contemplated.

The present invention also includes a cell, preferably a yeast cell transformed to express an albumin fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away. Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

Preferred yeast strains to be used in the production of albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1, pra1, ubc4] is a derivative of parent strain AH22his+ (also known as DB1; see, e.g., Sleep et al. Biotechnology 8:42-46 (1990)). The strain contains a leu2 mutation which allows for auxotropic selection of 2 micron-based plasmids that contain the LEU2 gene. D88 also exhibits a derepression of PRB1 in glucose excess. The PRB1 promoter is normally controlled by two checkpoints that monitor glucose levels and growth stage. The promoter is activated in wild type yeast upon glucose depletion and entry into stationary phase. Strain D88 exhibits the repression by glucose but maintains the induction upon entry into stationary phase. The PRA1 gene encodes a yeast vacuolar protease, YscA endoprotease A, that is localized in the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short lived and abnormal proteins for ubiquitin dependant degradation. Isolation of this ubc4 mutation was found to increase the copy number of an expression plasmid in the cell and cause an increased level of expression of a desired protein expressed from the plasmid (see, e.g., International Publication No. WO99/00504, hereby incorporated in its entirety by reference herein).

DXY1, a derivative of D88, has the following genotype: [leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to the mutations isolated in D88, this strain also has a knockout of the YAP3 protease. This protease causes cleavage of mostly di-basic residues (RR, RK, KR, KK) but can also promote cleavage at single basic residues in proteins. Isolation of this yap3 mutation resulted in higher levels of full length HSA production (see, e.g., U.S. Pat. No. 5,965,386, and Kerry-Williams et al., Yeast 14:161-169 (1998), hereby incorporated in their entireties by reference herein).

BXP10 has the following genotype: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition to the mutations isolated in DXY1, this strain also has a knockout of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of the evolutionarily conserved family of dolichyl-phosphate-D-mannose protein O-mannosyltransferases (Pmts). The transmembrane topology of Pmtlp suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in O-linked glycosylation. This mutation serves to reduce/eliminate O-linked glycosylation of HSA fusions (see, e.g., International Publication No. WO00/44772, hereby incorporated in its entirety by reference herein). Studies revealed that the Hsp150 protein is inefficiently separated from rHA by ion exchange chromatography. The mutation in the HSP150 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., U.S. Pat. No. 5,783,423, hereby incorporated in its entirety by reference herein.

The desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, i.e., cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which are described in detail in Example 2. FIG. 4 shows a map of the pPPC0005 plasmid that can be used as the base vector into which polynucleotides encoding Therapeutic proteins may be cloned to form HA-fusions. It contains a PRB1 S. cerevisiae promoter (PRB1p), a Fusion leader sequence (FL), DNA encoding HA (rHA) and an ADH1 S. cerevisiae terminator sequence. The sequence of the fusion leader sequence consists of the first 19 amino acids of the signal peptide of human serum albumin (SEQ ID NO:29) and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319 which is hereby incorporated by reference in its entirety.

The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were deposited on Apr. 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. Another vector useful for expressing an albumin fusion protein in yeast the pSAC35 vector which is described in Sleep et al., BioTechnology 8:42 (1990) which is hereby incorporated by reference in its entirety.

A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, γ-single-stranded termini with their 3′ 5′-exonucleolytic activities, and fill in recessed 3′-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA in accordance with the invention, if, for example, HA variants are to be prepared, is to use the polymerase chain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

Convenient regulatable promoters for use in Schizosaccharomyces pombe are the thiamine-repressible promoter from the nmt gene as described by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucose repressible jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.

The transcription termination signal is preferably the 3′ flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3′ flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADHI gene is preferred.

The desired albumin fusion protein may be initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen. Leaders useful in S. cerevisiae include that from the mating factor a polypeptide (MF α-1) and the hybrid leaders of EP-A-387 319. Such leaders (or signals) are cleaved by the yeast before the mature albumin is released into the surrounding medium. Further such leaders include those of S. cerevisiae invertase (SUC2) disclosed in JP 62-096086 (granted as 911036516), acid phosphatase (PH05), the pre-sequence of MFα-1, 0 glucanase (BGL2) and killer toxin; S. diastaticus glucoarnylase Il; S. carlsbergensis α-galactosidase (MEL1); K. lactis killer toxin; and Candida glucoanylase.

Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins

The present invention also relates to vectors containing a polynucleotide encoding an albumin fusion protein of the present invention, host cells, and the production of albumin fusion proteins by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides encoding albumin fusion proteins of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

In one embodiment, polynucleotides encoding an albumin fusion protein of the invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell. For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the albumin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs. In a specific embodiment, polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.

Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to, the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134), the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:34), and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:35). A suitable signal sequence that may be used in conjunction with baculoviral expression systems is the gp67 signal sequence (e.g., amino acids 1-19 of GenBank Accession Number AAA72759).

Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.

The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Therapeutic protein may be replaced with an albumin fusion protein corresponding to the Therapeutic protein), and/or to include genetic material (e.g., heterologous polynucleotide sequences such as for example, an albumin fusion protein of the invention corresponding to the Therapeutic protein may be included). The genetic material operably associated with the endogenous polynucleotide may activate, alter, and/or amplify endogenous polynucleotides.

In addition, techniques known in the art may be used to operably associate heterologous polynucleotides (e.g., polynucleotides encoding an albumin protein, or a fragment or variant thereof) and/or heterologous control regions (e.g., promoter and/or enhancer) with endogenous polynucleotide sequences encoding a Therapeutic protein via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose S100, S200, S300, superdex resin columns and their equivalents and comparables.

In specific embodiments the albumin fusion proteins of the invention are purified using Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are selective for either the HSA or the “fusion targef” molecules.

In preferred embodiments albumin fusion proteins of the invention are purified using one or more Chromatography methods listed above. In other preferred embodiments, albumin fusion proteins of the invention are purified using one or more of the following Chromatography columns, Q sepharose FF column, SP Sepharose FF column, Q Sepharose High Performance Column, Blue Sepharose FF column, Blue Column, Phenyl Sepharose FF column, DEAE Sepharose FF, or Methyl Column.

Additionally, albumin fusion proteins of the invention may be purified using the process described in International Publication No. WO00/44772 which is herein incorporated by reference in its entirety. One of skill in the art could easily modify the process described therein for use in the purification of albumin fusion proteins of the invention.

Albumin fusion proteins of the present invention may be recovered from: products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, albumin fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express albumin fusion proteins of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding an albumin fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

The invention encompasses albumin fusion proteins of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The albumin fusion proteins may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

In specific embodiments, albumin fusion proteins of the present invention or fragments or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177Lu, 90Y, 166Ho, and 153Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 90Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.

As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Albumin fusion proteins of the invention and antibodies that bind a Therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Techniques for conjugating such therapeutic moiety to proteins (e.g., albumin fusion proteins) are well known in the art.

Albumin fusion proteins may also be attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with albumin fusion proteins of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Albumin fusion proteins, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Also provided by the invention are chemically modified derivatives of the albumin fusion proteins of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The albumin fusion proteins may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a Therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the albumin fusion proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the albumin fusion protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (CISO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.

The presence and quantity of albumin fusion proteins of the invention may be determined using ELISA, a well known immunoassay known in the art. In one ELISA protocol that would be useful for detecting/quantifying albumin fusion proteins of the invention, comprises the steps of coating an ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or more different concentrations), adding a secondary anti-Therapeutic protein specific antibody coupled to a detectable label (as described herein or otherwise known in the art), and detecting the presence of the secondary antibody. In an alternate version of this protocol, the ELISA plate might be coated with the anti-Therapeutic protein specific antibody and the labeled secondary reagent might be the anti-human albumin specific antibody.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention. As described in more detail below, polynucleotides of the invention (encoding albumin fusion proteins) may be used in recombinant DNA methods useful in genetic engineering to make cells, cell lines, or tissues that express the albumin fusion protein encoded by the polynucleotides encoding albumin fusion proteins of the invention.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy”, and Examples 17 and 18).

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

Albumin fusion proteins of the invention can also be detected in vivo by imaging. Labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR) or electron spin relaxtion (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients given to a cell line expressing the albumin fusion protein of the invention.

An albumin fusion protein which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc, (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133 Xe), fluorine (18F, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at locations in the body (e.g., organs, cells, extracellular spaces or matrices) where one or more receptors, ligands or substrates (corresponding to that of the Therapeutic protein used to make the albumin fusion protein of the invention) are located. Alternatively, in the case where the albumin fusion protein comprises at least a fragment or variant of a Therapeutic antibody, the labeled albumin fusion protein will then preferentially accumulate at the locations in the body (e.g., organs, cells, extracellular spaces or matrices) where the polypeptides/epitopes corresponding to those bound by the Therapeutic antibody (used to make the albumin fusion protein of the invention) are located. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)). The protocols described therein could easily be modified by one of skill in the art for use with the albumin fusion proteins of the invention.

In one embodiment, the invention provides a method for the specific delivery of albumin fusion proteins of the invention to cells by administering albumin fusion proteins of the invention (e.g., polypeptides encoded by polynucleotides encoding albumin fusion proteins of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering albumin fusion proteins of the invention in association with toxins or cytotoxic prodrugs.

By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as, for example, 103Pd, 133Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169b, 51Cr, 54Mn, 75Se, 113Sn, 90Yttrium, 117Tin, 186Rhenium, 166Holmium, and 188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131I.

Techniques known in the art may be applied to label polypeptides of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).

The albumin fusion proteins of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein under the section heading “Biological Activities,” below.

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using an albumin fusion protein of the invention; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, albumin fusion proteins of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

In particular, albumin fusion proteins comprising of at least a fragment or variant of a Therapeutic antibody can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can bind, and/or neutralize the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds, and/or reduce overproduction of the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds. Similarly, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can activate the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds, by binding to the polypeptide bound to a membrane (receptor).

At the very least, the albumin fusion proteins of the invention of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Albumin fusion proteins of the invention can also be used to raise antibodies, which in turn may be used to measure protein expression of the Therapeutic protein, albumin protein, and/or the albumin fusion protein of the invention from a recombinant cell, as a way of assessing transformation of the host cell, or in a biological sample. Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.

For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding a polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.

The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome

By “assaying the expression level of the gene encoding a polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of a particular polypeptide (e.g. a polypeptide corresponding to a Therapeutic protein disclosed in Table 1) or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).

The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting abnormal expression of polypeptides that bind to, are bound by, or associate with albumin fusion proteins compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide that bind to, are bound by, or associate with albumin fusion proteins of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.

Assaying polypeptide levels in a biological sample can occur using a variety of techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of interest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.

For example, albumin fusion proteins may be used to quantitatively or qualitatively detect the presence of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the present invention. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytometric, or fluorimetric detection.

In a preferred embodiment, albumin fusion proteins comprising at least a fragment or variant of an antibody that immunospecifically binds at least a Therapeutic protein disclosed herein (e.g., the Therapeutic proteins disclosed in Table 1) or otherwise known in the art may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.

The albumin fusion proteins of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of polypeptides that bind to, are bound by, or associate with an albumin fusion protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The albumin fusion proteins are preferably applied by overlaying the labeled albumin fusion proteins onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the polypeptides that bind to, are bound by, or associate with albumin fusion proteins, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays that detect polypeptides that bind to, are bound by, or associate with albumin fusion proteins will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled albumin fusion protein of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable of binding a polypeptide (e.g., an albumin fusion protein, or polypeptide that binds, is bound by, or associates with an albumin fusion protein of the invention.) Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polypeptide. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of albumin fusion protein may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

In addition to assaying polypeptide levels in a biological sample obtained from an individual, polypeptide can also be detected in vivo by imaging. For example, in one embodiment of the invention, albumin fusion proteins of the invention are used to image diseased or neoplastic cells.

Labels or markers for in vivo imaging of albumin fusion proteins of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients of a cell line (or bacterial or yeast strain) engineered.

Additionally, albumin fusion proteins of the invention whose presence can be detected, can be administered. For example, albumin fusion proteins of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.

A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at the locations in the body which contain a polypeptide or other substance that binds to, is bound by or associates with an albumin fusion protein of the present invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

One of the ways in which an albumin fusion protein of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Albumin fusion proteins may also be radiolabelled and used in any of a variety of other immunoassays. For example, by radioactively labeling the albumin fusion proteins, it is possible to the use the albumin fusion proteins in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

It is also possible to label the albumin fusion proteins with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The albumin fusion protein can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The albumin fusion proteins can also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged albumin fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label albumin fusion proteins of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Transgenic Organisms

Transgenic organisms that express the albumin fusion proteins of the invention are also included in the invention. Transgenic organisms are genetically modified organisms into which recombinant, exogenous or cloned genetic material has been transferred. Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene may include one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, that may be necessary for optimal expression and secretion of the encoded protein. The transgene may be designed to direct the expression of the encoded protein in a manner that facilitates its recovery from the organism or from a product produced by the organism, e.g. from the milk, blood, urine, eggs, hair or seeds of the organism. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal. The transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.

The term “germ cell line transgenic organism” refers to a transgenic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic organisms. The alteration or genetic information may be foreign to the species of organism to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

A transgenic organism may be a transgenic animal or a transgenic plant. Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Pat. No. 5,489,743 and U.S. Pat. No. 5,602,307.

To direct the secretion of the transgene-encoded protein of the invention into the milk of transgenic mammals, it may be put under the control of a promoter that is preferentially activated in mammary epithelial cells. Promoters that control the genes encoding milk proteins are preferred, for example the promoter for casein, beta lactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio (1992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology 7:487492; Gorton et al. (1987) BioTechnology 5:1183-1187; and Soulier et al. (1992) FEBS Letts. 297:13). The transgenic mammals of choice would produce large volumes of milk and have long lactating periods, for example goats, cows, camels or sheep.

An albumin fusion protein of the invention can also be expressed in a transgenic plant, e.g. a plant in which the DNA transgene is inserted into the nuclear or plastidic genome. Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art (e.g., see Example 19). See, in general, Methods in Enzymology Vol. 153 (“Recombinant DNA Part D”) 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554. Methods for generation of genetically engineered plants are further described in U.S. Pat. No. 5,283,184, U.S. Pat. No. 5,482,852, and European Patent Application EP 693 554, all of which are hereby incorporated by reference.

Pharmaceutical or Therapeutic Compositions

The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral (e.g. subcutaneous or intramuscular) injection or intravenous infusion. The treatment may consist of a single dose or a plurality of doses over a period of time.

While it is possible for an albumin fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be “acceptable” in the sense of being compatible with the albumin fusion protein and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. Albumin fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf-life in solution. For instance, pharmaceutical compositions of the invention may be formulated well in advance in aqueous form, for instance, weeks or months or longer time periods before being dispensed.

For example, wherein the Therapeutic protein is hGH, EPO, alpha-IFN or beta-IFN, formulations containing the albumin fusion protein may be prepared taking into account the extended shelf-life of the albumin fusion protein in aqueous formulations. As exhibited in Table 2, most Therapeutic proteins are unstable with short shelf-lives after formulation with an aqueous carrier. As discussed above, the shelf-life of many of these Therapeutic proteins are markedly increased or prolonged after fusion to HA.

TABLE 2

Tradename,

Storage Conditions of

Protein

Manufacturer

Route

Formulation

Non-Fusion Protein

Interferon,

Roferon-A,

sc

sol_n

4-8° C.

alpha-2a

Hoffmann-LaRoche

im

(vial or pre-filled

syringe)

Interferon,

Intron-A,

iv sc im

sol_n;

4-8° C.

alpha-2b

Schering Plough

powder + dil.

(all preps, before and after

dilution)

COMBO

Rebetron (Intron-A +

po +

Rebetol capsule +

Interferon alpha-

Rebetol)

sc

Intron-A injection

2b +

Schering Plough

Ribavirin

Interferon,

Infergen

sc

sol_n

4-8° C.

Alphacon-1

Amgen

Interferon,

Wellferon,

sc

sol_n

4-8° C.

alpha-n1,

Wellcome

im

(with albumin _as

Lympho-

stablizer_)

blastoid

Interferon,

Avonex,

im

powder + dil.

4-8° C.

beta-1a

Biogen

(with albumin)

(before and after dilution)

(Use within 3-6 h of

reconstitution)

Rebif,

sc

sol_n,

Ares-Serono

in pre-filled syringe

(Europe only)

Interferon,

Betaseron,

sc

powder + dil.

4-8° C.

beta-1b

Chiron

(with albumin)

(before and after dilution)

(Europe: Betaferon)

(Use within 3 h of

reconstitution)

Single use vials.

Interferon,

Actimmune,

sc

4-8° C.

Gamma-1b

InterMune

(before and after dilution)

Pharmaceuticals

(Use within 3 h of

reconstitution).

Growth

Genotropin,

powder/dil cartridges

4-8° C.

Hormone

Pharmacia Upjohn

(single or multi-use);

(before and after dilution);

(somatropin)

single use MiniQuick

single use MiniQuick

injector

Delivery Device should

be refrigerated until use.

Humatrope,

sc

powder + dil.

4-8° C.

Eli Lilly

im

(Vial or pen cartridge)

(before and after dilution)

(Use vials within 25 h,

cartridges within 28 d, of

reconstitution).

Norditropin,

Novo Nordisk

Pharmaceuticals

Nutropin,

sc

powder + dil.

4-8° C.

Genentech

(stable for 14 d after dil_n)

(all preps, before and after

dilution)

Nutropin AQ,

sc

sol_n

4-8° C.

Genentech

(Stable for 28 d after 1st

use)

Nutropin Depot,

sc

microsphere

4-8° C.

Genentech

suspension as

Single use pkges. Dose

powder + dil.

1-2x/month (ProLease

micro-encapsulation

technol.)

Saizen,

sc

powder + dil.

Powder _should be stored

(Serono)

im

at Rm Temp_. After

reconstitution store 4-

8° C. for up to 14 d.

Serostim,

Powder _should be stored

Serono

at Rm Temp_. After

reconstitution store in 4-

8° C. for up to 14 d.

hGH, with

Protropin,

sc

powder + dil.

4-8° C.

N-term. Met

Genentech

im

(all preps, before and

(somatrem)

after dilution)

Erythropoietin

Epogen,

iv

sol_n

4-8° C.

(Epoetin alfa)

Amgen

sc

(use within 21 d of first

use)

(Single & multi-dose

vials)

Procrit,

iv

sol_n

4-8° C.

Amgen

sc

(use within 21 d of first

use)

(Single & multi-dose

vials)

In instances where aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as aerosols using standard procedures. The term “aerosol” includes any gas-borne suspended phase of an albumin fusion protein of the instant invention which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets of an albumin fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al,. (1992) Pharmacol. Toxicol. Methods 27:143-159.

The formulations of the invention are also typically non-immunogenic, in part, because of the use of the components of the albumin fusion protein being derived from the proper species. For instance, for human use, both the Therapeutic protein and albumin portions of the albumin fusion protein will typically be human. In some cases, wherein either component is non human-derived, that component may be humanized by substitution of key amino acids so that specific epitopes appear to the human immune system to be human in nature rather than foreign.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the albumin fusion protein with the carrier that constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation appropriate for the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders. Dosage formulations may contain the Therapeutic protein portion at a lower molar concentration or lower dosage compared to the non-fused standard formulation for the Therapeutic protein given the extended serum half-life exhibited by many of the albumin fusion proteins of the invention.

As an example, when an albumin fusion protein of the invention comprises growth hormone as one or more of the Therapeutic protein regions, the dosage form can be calculated on the basis of the potency of the albumin fusion protein relative to the potency of hGH, while taking into account the prolonged serum half-life and shelf-life of the albumin fusion proteins compared to that of native hGH. Growth hormone is typically administered at 0.3 to 30.0 IU/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three or seven divided doses for a year or more. In an albumin fusion protein consisting of full length HA fused to full length GH, an equivalent dose in terms of units would represent a greater weight of agent but the dosage frequency can be reduced, for example to twice a week, once a week or less.

Formulations or compositions of the invention may be packaged together with, or included in a kit with, instructions or a package insert referring to the extended shelf-life of the albumin fusion protein component. For instance, such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the extended or prolonged shelf-life of the albumin fusion proteins of the invention. Such instructions or package inserts may also address the particular advantages of the albumin fusion proteins of the inventions, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions. As described above, formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.

Albumin fusion proteins of the invention can also be included in nutraceuticals. For instance, certain albumin fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses albumin fusion protein. Such compositions can also include plant or plant products obtained from a transgenic plant which expresses the albumin fusion protein. The albumin fusion protein can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, December 1993.

The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of an albumin fusion protein of the invention or a polynucleotide encoding an albumin fusion protein of the invention (“albumin fusion polynucleotide”) in a pharmaceutically acceptable carrier.

The albumin fusion protein and/or polynucleotide will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the albumin fusion protein and/or polynucleotide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the albumin fusion protein administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the albumin fusion protein is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 14 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Albumin fusion proteins and/or polynucleotides can be are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the albumin fusion protein and/or polynucleotide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the albumin fusion protein and/or polynucleotide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The albumin fusion protein is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Albumin fusion proteins and/or polynucleotides generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Albumin fusion proteins and/or polynucleotides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or polynucleotide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized albumin fusion protein and/or polynucleotide using bacteriostatic Water-for-Injection.

In a specific and preferred embodiment, the Albumin fusion protein formulations comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In another specific and preferred embodiment, the Albumin fusion protein formulations consists 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. The pH and buffer are chosen to match physiological conditions and the salt is added as a tonicifier. Sodium octanoate has been chosen due to its reported ability to increase the thermal stability of the protein in solution. Finally, polysorbate has been added as a generic surfactant, which lowers the surface tension of the solution and lowers non-specific adsorption of the albumin fusion protein to the container closure system.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the albumin fusion proteins and/or polynucleotides of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the albumin fusion proteins and/or polynucleotides may be employed in conjunction with other therapeutic compounds.

The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparations of Corynebacterium parvum. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with alum. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with other therapeutic agents. Albumin fusion protein and/or polynucleotide agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.

In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKINASE™), antiresplace (e.g., EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICAR™). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.

In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).

In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEP™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with albumin fusion proteins and/or polynucleotides of the invention to treat AIDS and/or to prevent or treat HIV infection.

Additional NNRTIs include COACTINON™ (Emivirine/MKC442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (see, International Publication No. WO 99/49830).

In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In other embodiments, albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunestimulants. Immunostimulants that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).

In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not lmited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).

In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.

In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.)

In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.

In another specific embodiment, the compositions of the invention are administered in combination Zevalin™. In a further embodiment, compositions of the invention are administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Particularly preferred isotopes are 90Y and 111In.

In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with cytokines. Cytokines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.

In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In additional embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising albumin fusion proteins of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Gene Therapy

Constructs encoding albumin fusion proteins of the invention can be used as a part of a gene therapy protocol to deliver therapeutically effective doses of the albumin fusion protein. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, encoding an albumin fusion protein of the invention. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous nucleic acid molecules encoding albumin fusion proteins in vivo. These vectors provide efficient delivery of nucleic acids into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:27 1). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.

Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., cited supra; Haj-Ahmand et al., J. Virol. 57:267 (1986)).

In another embodiment, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject nucleotide molecule by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. In a representative embodiment, a nucleic acid molecule encoding an albumin fusion protein of the invention can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-5 5 1; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A43075).

Gene delivery systems for a gene encoding an albumin fusion protein of the invention can be introduced into a patient by any of a number of methods. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 05 7). The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Where the albumin fusion protein can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the albumin fusion protein.

Additional Gene Therapy Methods

Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of an albumin fusion protein of the invention. This method requires a polynucleotide which codes for an albumin fusion protein of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the fusion protein of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J. -F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, polynucleotides encoding the albumin fusion proteins of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, polynucleotides encoding the albumin fusion proteins of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOIMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding an albumin fusion protein of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a fusion protin of the present invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses fusion protein of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a fsuion protein of the invention.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotide encoding an albumin fusion protein of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, an albumin fusion protein of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, include fusion proteins of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising albumin fusion proteins of the invention for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.

Albumin fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.

Biological Activities

Albumin fusion proteins and/or polynucleotides encoding albumin fusion proteins of the present invention, can be used in assays to test for one or more biological activities. If an albumin fusion protein and/or polynucleotide exhibits an activity in a particular assay, it is likely that the Therapeutic protein corresponding to the fusion portein may be involved in the diseases associated with the biological activity. Thus, the fusion protein could be used to treat the associated disease.

Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, albumin fusion proteins of the invention and polynucleotides encoding these protiens, may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.

In a preferred embodiment, albumin fusion proteins of the invention comprising a Therapeutic protein portion corresponding to B-glucocerebrosidase and/or fragments or variants thereof can be used to treat, prevent, diagnose, prognose, and/or detect Gaucher's disease and/or as described under “Blood Related Disorders” and/or “Hyperproliferative Disorders” infra.

In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indication Y” column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein disclosed in the “Therapeutic Protein X” column of Table 1 (in the same row as the disease or disorder to be treated is listed in the “Preferred Indication Y” column of Table 1) in an amount effective to treat, prevent or ameliorate the disease or disorder.

In certain embodiments, an albumin fusion protein of the present invention may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the gene corresponding to the Therapeutic protein portion of the fusion portien of the invention is expressed.

Thus, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.

More generally, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful for the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with the following systems.

Immune Activity

Albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular immune system disease or disorder.

In another embodiment, a fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed.

In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.

In a preferred embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.

The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.

In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a immunosuppressive agent(s).

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.

Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate IgE concentrations in vitro or in vivo.

In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance tumor-specific immune responses.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.

In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell responsiveness to pathogens.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an activator of T cells.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to induce higher affinity antibodies.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to increase serum immunoglobulin concentrations.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to accelerate recovery of immunocompromised individuals.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in the pretreatment of bone marrow samples prior to transplant.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.

The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit complement mediated cell lysis.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed to treat adult respiratory distress syndrome (ARDS).

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to stimulate the regeneration of mucosal surfaces.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.

In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.

Blood-Related Disorders

The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.

In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed.

The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias described below. Alternatively, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.

The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, treat, or diagnose blood dyscrasia.

The effect of the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.

Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.

In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.

Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.

In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a treatment prior to surgery, to increase blood cell production.

In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.

In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.

In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase cytokine production.

In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.

Hyperproliferative Disorders

In certain embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.

In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose and/or prognose disorders associated with the tissue(s) in which the polypeptide of the invention is expressed.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to, those described herein. In a further preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.

In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleofides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferafive disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide encoding an albumin fusion protein of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the fusion protein of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference).

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. These fusion protieins and/or polynucleotides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and −2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, these fusion proteins and/or polynucleotides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of these proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. April 24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporated by reference).

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering these albumin fusion proteins and/or polynucleotides, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to targeted cells expressing the a polypeptide bound by, that binds to, or associates with an albumin fuison protein of the invention. Albumin fusion proteins of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Albumin fusion proteins of the invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the albumin fusion proteins of the invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Renal Disorders

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.

Compositions of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Compositions of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.

Cardiovascular Disorders

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Methods of delivering polynucleotides are described in more detail herein.

Respiratory Disorders

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.

The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.

It is believed that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may have a cytoprotective effect on the small intestine mucosa. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat diseases associate with the under expression.

Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neural Activity and Neurological Diseases

The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In one embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.

In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.

In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a heart atta